Human monoclonal antibodies to human nucleolin

ABSTRACT

The present invention provides for methods of producing human monoclonal antibodies to human nucleolin, cells producing such antibodies, and the antibodies themselves. Also provided are methods of using the antibodies in diagnosing and treating malignant and non-malignant diseases wherein cells that express nucleolin on the cell surface contribute to the pathophysiology of the disease.

CROSS-REFERENCE

This application is a national phase application under 35 U.S.C. 371 ofInternational Application No. PCT/US2010/057046 filed Nov. 17, 2010which claims benefit of priority to U.S. Provisional Application Ser.No. 61/261,909, filed Nov. 17, 2009, which is incorporated herein byreference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder grant number NCI CA109254-04S1 awarded by the National CancerInstitute, National Institutes of Health. The U.S. Government may haverights in this invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of cell biologyand immunology. More particularly, it concerns methods and compositionsrelating to the production and use of human monoclonal antibodies tohuman nucleolin (NCL).

Antibodies are a class of agents known as “biologicals.” The source ofantibodies can be a polyclonal supply, such as human or horse serum, orderived from a monoclonal source (single cell clone). With thetechnologic capability to control and select for specific antigenbinding, monoclonal antibodies have yielded dramatic therapeuticbenefits. However, the difficulty of generating specific antibodies forcertain targets has limited the successes, and the potential fortherapeutic and diagnostic agents remains largely untapped.

One impediment to the development of monoclonal antibodies for humantherapy is the need to “humanize” such antibodies, which are generallymade in mice, rats and rabbits. If human patients are administered suchantibodies without humanized constant regions, they can suffer from“serum sickness,” meaning that an endogenous immune response is mountedby the recipient against the non-human antibody sequences. Humanizingmonoclonal antibodies produced in research animals can avoid thisproblem. However, the cost in time and expense for humanization ofantibodies can be considerable.

Nucleolin is expressed on the cell surface of human chronic lymphocyticleukemia (CLL) cells, acute myeloid leukemia (AML) cells, some forms ofbreast carcinoma, as well as other tumors. As such, nucleolinconstitutes a promising tumor antigen for targeting of therapeutics,including antibodies.

SUMMARY OF THE INVENTION

In one aspect of the invention a method is provided of producing animmortalized human B-cell that secrets an antibody that binds to humannucleolin comprising: obtaining a population of IgM-positive humanB-cells; contacting said population with: Epstein-Barr virus (EBV) toimmortalize said human B-cells, and a cytokine/growth factor/signalingagent cocktail to induce IgM-to-IgG immunoglobulin isotypeclass-switching; and culturing cells under conditions supporting saidimmortalization and immunoglobulin isotype class-switching. In oneembodiment the method further comprises, selecting an immortalized humanB-cell that expresses an antibody to human nucleolin. In anotherembodiment, selecting comprises an immunoassay performed on immortalizedB-cell culture medium supernatants. In another embodiment, a cytokinecocktail comprises an agent that delivers a costimulatory signal to ahuman B-cell. In another embodiment, a cytokine cocktail comprisesanti-IgM F(ab′)₂ interleukin (IL)-2, IL-4, IL-5, IL-6, IL-9, IL-10,IL-13, INFα, BAFF, soluble CD40L. In another embodiment, a population ofIgM-positive human B-cells is obtained from peripheral blood, a tonsilsbone marrow, a spleen, a lymph node, umbilical cord blood, a liver, anapheresis procedures or a buffy coat. In another embodiment, the methodfurther comprises isolating a nucleic acid encoding an entire heavyand/or light chain from the immortalized human B-cell of step (d). Inanother embodiment, the method further comprises isolating a nucleicacid encoding a heavy and/or light chain antigen-binding region from theimmortalized human B-cell of step (d). In another embodiment, the methodfurther comprises cloning said nucleic acid into a nucleic acid encodinga framework region of a heavy and/or light chain. In another embodiment,contacting said population further comprises an EBV concentration step,a centrifugation step during infection, or both. In another embodiment,the method further comprises freezing said population of human B-cellsfollowing step (c). In another embodiment, contacting said populationwith a cytokine/growth factor/signaling agent cocktail is performed atabout 0-96 hours following step (b)(ii). In another embodiment,contacting said population with a cytokine/growth factor/signaling agentcocktail is performed at about 16-20 hours following step (b)(ii). Inanother embodiment, about 50%-99% of said population are immortalized byEBV infection. In another embodiment, about 95%-99% of said populationare immortalized by EBV infection. In another embodiment, selecting animmortalized human B-cell that expresses an antibody to human nucleolinoccurs 1-4 weeks following infection. In another embodiment, selectingan immortalized human B-cell that expresses an antibody to humannucleolin occurs 2-3 weeks following infection. In another embodiment,selecting an immortalized human B-cell that expresses an antibody tohuman nucleolin occurs after thawing stored frozen immortalized B-cells,and/or after thawing stored frozen culture medium supernatants from saidimmortalized B-cells. In another embodiment, the B-cell is antigennaïve. In another embodiment, the B-cell is antigen experienced.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody that binds to human nucleolin. In oneembodiment, the immortalized human B-cell is designated as T-5D1, V-3H11(3G5), T-2D3, T-7G7 (1H9), T-2H3, T-9F9, T-8G4 or T-P1C6.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein of SEQ ID No. 2. In one embodiment, the IgG antibody is an IgG1,IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody isan IgG1 antibody. In another embodiment, the IgG antibody comprises akappa light chain. In another embodiment, the IgG antibody comprises alambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 10% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein encoded by SEQ ID No. 1. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. The isolated antibody orfragment thereof of claim 33, wherein said antibody or fragment thereofis substantially non-immunogenic to a human. In another embodiment, theantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the antibody or fragment thereof kills at least 10%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 20% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 30%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 40% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 50%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 60% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 70%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 80% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 90%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 100% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 10%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 20% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 30%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 40% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 50%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 60% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 70%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 80% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 90%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 100% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof binds to an RNAbinding domain of human nucleolin. In another embodiment, the antibodyor fragment thereof inactivates an RNA binding domain of humannucleolin. In another embodiment, the isolated antibody or fragmentthereof induces complement-dependent cytotoxicity to a cancer cell. Inanother embodiment, the isolated antibody or fragment thereof inducescomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the isolatedantibody or fragment thereof inhibits or kills an AML cancer cell, a CLLcancer cell or a breast cancer cell. In another embodiment, the isolatedmonoclonal antibody or fragment thereof reduces BCL-2 levels in a cancercell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein comprising SEQ ID No. 4. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the isolated antibody or fragmentthereof of claim 33, wherein said antibody or fragment thereof is amonoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 10% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein encoded by SEQ ID No. 3. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 20% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated human monoclonalantibody or fragment thereof that specifically binds to a protein of SEQID No. 4. In one embodiment, the antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated antibody orfragment thereof that specifically binds to a human nucleolin protein,wherein said antibody or fragment thereof kills at least 20% of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In one embodiment, the amino acid sequenceof said human nucleolin comprises SEQ ID No. 2. In another embodiment,the antibody or fragment thereof binds to an amino acid sequenceconsisting of amino acid residues 1 to 283 of SEQ ID No. 2. In anotherembodiment, the antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof binds to an RNA binding domain of human nucleolin. In anotherembodiment, the antibody or fragment thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the isolated antibodyor fragment thereof is linked to a diagnostic or therapeutic agent. Inanother embodiment, the isolated antibody or fragment thereof exhibitscomplement-dependent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof exhibitscomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the antibody orfragment thereof inhibits or kills an AML cancer cell, a CLL cancer cellor a breast cancer cell. In another embodiment, the isolated antibody orfragment thereof reduces BCL-2 levels in a cancer cell. In anotherembodiment, the antibody or fragment thereof is linked to a diagnosticagent. In another embodiment, the diagnostic agent is a radionuclide, afluorophore, a chemilluminescent compound, a fluorescent compound, or anenzyme. In another embodiment, the antibody or fragment thereof islinked to a therapeutic agent. In another embodiment, the therapeuticagent is a radionuclide, a toxin or a chemotherapeutic moiety.

In another aspect the invention provides an isolated antibody orfragment thereof that specifically binds to a human nucleolin protein,wherein said antibody or fragment thereof kills at least 10-100% of apopulation of MV4-11 cells (such as 10, 20, 30, 40, 50, 60, 70, 80, 90or 100% of a population of MV4-11 cells, when incubated with said MCF-7cells and human AB serum for 48 hours. In another embodiment, the aminoacid sequence of said human nucleolin comprises SEQ ID No. 2. In anotherembodiment, the antibody or fragment thereof binds to an amino acidsequence consisting of amino acid residues 1 to 283 of SEQ ID No. 2. Inanother embodiment, the antibody or fragment thereof is a monoclonalantibody or fragment thereof. In another embodiment, the antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof binds to an RNA binding domain of human nucleolin. In anotherembodiment, the antibody or fragment thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the isolated antibodyor fragment thereof is linked to a diagnostic or therapeutic agent. Inanother embodiment, the isolated antibody or fragment thereof exhibitscomplement-dependent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof exhibitscomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the antibody orfragment thereof inhibits or kills an AML cancer cell, a CLL cancer cellor a breast cancer cell. In another embodiment, the isolated antibody orfragment thereof reduces BCL-2 levels in a cancer cell. In anotherembodiment, the said antibody or fragment thereof is linked to adiagnostic agent. In another embodiment, the diagnostic agent is aradionuclide, a fluorophore, a chemilluminescent compound, a fluorescentcompound, or an enzyme. In another embodiment, the antibody or fragmentthereof is linked to a therapeutic agent. In another embodiment, thetherapeutic agent is a radionuclide, a toxin or a chemotherapeuticmoiety.

In another aspect the invention provides an anti-nucleolin compositioncomprising one or more isolated antibodies or fragments thereof thatspecifically binds to a human nucleolin protein, wherein said one ormore antibodies kills at least 10-100% of a population of MCF-7 cells(such as 10, 20, 30, 40, 40, 50, 60, 70, 80, 90, or 100%), whenincubated with said MCF-7 cells and human AB serum for 48-96 (such as48, 72 or 96 hours) hours. In one embodiment, the one or more isolatedantibodies or fragments thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the one or more isolated antibodies orfragments thereof is substantially non-immunogenic to a human. Inanother embodiment, the one or more isolated antibodies or fragmentsthereof is a human antibody or fragment thereof. In another embodiment,the one or more isolated antibodies or fragments thereof binds to an RNAbinding domain of human nucleolin. In another embodiment, the one ormore isolated antibodies or fragments thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the amino acidsequence of said human nucleolin comprises SEQ ID No. 2. In anotherembodiment, the one or more isolated antibodies or fragments thereofbinds to SEQ ID No. 4. In another embodiment, the anti-nucleolincomposition further comprises a radionuclide, a fluorophore, achemilluminescent compound, a fluorescent compound, an enzyme, a toxinor a chemotherapeutic agent. In another embodiment, the radionuclide, afluorophore, a chemilluminescent compound, a fluorescent compound, anenzyme, a toxin or a chemotherapeutic agent is conjugated to said one ormore isolated antibodies or fragments thereof. In another embodiment,the anti-nucleolin composition comprises two or more isolated antibodiesor fragments thereof that specifically binds to said human nucleolinprotein, wherein said one or more antibodies kills at least 20% of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the anti-nucleolincomposition comprises three or more isolated antibodies or fragmentsthereof that specifically binds to said human nucleolin protein, whereinsaid one or more antibodies kills at least 20% of a population of MCF-7cells, when incubated with said MCF-7 cells and human AB serum for 48hours.

In another aspect the invention provides a method of inhibiting orkilling a cell expressing nucleolin on its surface comprising contactingsaid cell with an antibody or fragment thereof that binds to humannucleolin, wherein said antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In one embodiment, the antibody or fragmentthereof is a human antibody or fragment thereof. In another embodiment,the antibody or fragment thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the antibody or fragment thereof bindsto SEQ ID No. 2. In another embodiment, the antibody or fragment thereofbinds to an amino acid sequence encoded by SEQ ID No. 1. In anotherembodiment, the antibody or fragment thereof binds to SEQ ID No. 4. Inanother embodiment, the antibody or fragment thereof binds to an aminoacid sequence encoded by SEQ ID No. 3. In another embodiment, the cellis a cancer cell. In another embodiment, the cancer cell is a lungcancer cell, a breast cancer cell, a prostate cancer cell, a coloncancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, anovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastomacell, a neuroblastoma cell, a sarcoma cell or a gastric cancer cell. Inanother embodiment, the cell is an immune cell. In another embodiment,the immune cell is a lymphocyte, dendritic cell, a peripheral bloodmonocyte, a macrophage or a glial cell. In another embodiment, theimmune cell is an activated immune cell. In another embodiment, theimmune cell is an activated B cell. In another embodiment, the immunecell is a memory B cell. In another embodiment, the immune cell is anactivated T cell. In another embodiment, the immune cell is an activatedCD4+ T cell. In another embodiment, the immune cell is an activated CD8+T cell. In another embodiment, the cell a vascular smooth muscle cell oran endothelial cell. In another embodiment, the antibody or fragmentthereof is linked to a therapeutic agent. In another embodiment, thetherapeutic agent is a radionuclide, a toxin or a chemotherapeuticagent. In another embodiment, the inhibiting or killing comprisesinducing apoptosis in said cell. In another embodiment, the cell islocated in a human subject, and said contacting comprising administeringsaid antibody or fragment thereof to said subject. In anotherembodiment, the method of further comprises contacting said cell with atleast one additional inhibitory agent or treatment. In anotherembodiment, the additional treatment comprises one or more of surgery,radiotherapy, chemotherapy, toxin therapy, immunotherapy, hormonetherapy, anti-angiogenic therapy or gene therapy or other biologicaltherapies. In another embodiment, the additional inhibitory agentcomprises one or more of radionuclides, chemotherapetic agents, toxinsimmunotherapeutics, hormones, nucleic acids or polypeptides. In anotherembodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin Achain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, phytolaca americana protein, pokeweedantiviral protein, momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin, calicheamicins or tricothecenes toxin. In anotherembodiment, the chemotherapeutic agent is an alkylating agent,anthracycline, cytoskeletal disruptor, epothilone, inhibitor oftopoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotideanalog, precursor analogs, peptide antibiotic, platinum-based agentsretinoids, vinca alkaloids or derivatives thereof. In anotherembodiment, the wherein said chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of detecting a cellexpressing nucleolin on its surface comprising contacting said cell witha human antibody or fragment thereof that binds immunologically to saidnucleolin. In one embodiment, the cell is a cancer cell, an immune cell,or a vascular smooth muscle cell that expresses nucleolin on itssurface, an endothelial cell that expresses nucleolin on its surface, ora virus infected cell. In another embodiment, the cell is a precancerouscell that expresses nucleolin on its surface. In another embodiment, thecancer cell selected from the group consisting of is a lung cancer cell,a breast cancer cell, a prostate cancer cell, a colon cancer cell, apancreatic cancer cell, a renal cell carcinoma cell, an ovarian cancercell, a leukemia cell, a melanoma cell, a glioblastoma cell, aneuroblastoma cell, a sarcoma cell and a gastric cancer cell. In anotherembodiment, the immune cell is a lymphocyte, dendritic cell, aperipheral blood monocyte, a macrophage and a glial cell. In anotherembodiment, the cell is an immune cell. In another embodiment, theimmune cell is an activated immune cell. In another embodiment, theimmune cell is an activated B cell. In another embodiment, the immunecell is a memory B cell. In another embodiment, the immune cell is anactivated T cell. In another embodiment, the immune cell is an activatedCD4+ T cell. In another embodiment, the immune cell is an activated CD8+T cell. In another embodiment, the cell is a vascular smooth muscle cellor an endothelial cell. In another embodiment, the antibody or fragmentthereof is linked to a diagnostic agent. In another embodiment, thediagnostic agent is a radionuclide, a fluorophore, a chemilluminescentcompound, a fluorescent compound, a quantum dot, a nanoparticles or anenzyme. In another embodiment, the cell is located in a human subjectand contacting comprises administering said antibody or fragment thereofto said subject. In another embodiment, the cell is located in anisolated, tissue sample or cell suspension.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (such as 10, 20, 30,40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 (such as 48,72, or 96) hours. In one embodiment, the mammal is a human. In anotherembodiment, the treating cancer in a mammal comprises treating tumorhpoxia. In another embodiment, the treating cancer in a mammal comprisesinhibiting tumor angiogenesis. In another embodiment, the anti-nucleolinantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is a human antibody or fragment thereof. In another embodiment,the anti-nucleolin monoclonal antibody or fragment thereof is amonoclonal antibody or fragment thereof. In another embodiment, thetoxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, phytolaca americana protein, pokeweed antiviralprotein, momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, calicheamicins or tricothecenes toxin. In another embodiment,the chemotherapeutic agent is an alkylating agent, anthracycline,cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I,inhibitor of topoisomerase II, nucleoside or nucleotide analog,precursor analogs, peptide antibiotic, platinum-based agents retinoids,vinca alkaloids or derivatives thereof. In another embodiment, thechemotherapeutic agent is actinomycin-D, all-trans retinoic acidazacitidine, adriamycin azathioprine, bleomycin, camptothecin,carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide,cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin,epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil (5FU),gemcitabine, hydroxyurea, hydrogen peroxide, idarubicin, imatinib,mechlorethamine, mercaptopurine, methotrexate, mitomycin C,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,tioguanine, valrubicin, vinblastine, vincristine, vindesine, orvinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin antibody orfragment thereof, a toxin or chemotherapeutic agent and apharmaceutically acceptable carrier, wherein said antibody or fragmentthereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% of a population of MCF-7 cells, when incubated with saidMCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. Inone embodiment, the mammal is a human. In another embodiment, thetreating cancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin antibody orfragment thereof and a pharmaceutically acceptable carrier, and furthertreating said mammal with radiation therapy, wherein said antibody orfragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60,70, 80, 90, or 100% of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96)hours. In one embodiment, the mammal is a human. In another embodiment,the treating cancer in a mammal comprises treating tumor hpoxia. Inanother embodiment, the treating cancer in a mammal comprises inhibitingtumor angiogenesis. In another embodiment, the anti-nucleolin antibodyor fragment thereof is substantially non-immunogenic to a human. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is a monoclonalantibody or fragment thereof. In another embodiment, the toxin isdiphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, phytolaca americana protein, pokeweed antiviral protein,momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin,calicheamicins or tricothecenes toxin. In another embodiment, thechemotherapeutic agent is an alkylating agent, anthracycline,cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I,inhibitor of topoisomerase H, nucleoside or nucleotide analog, precursoranalogs, peptide antibiotic, platinum-based agents retinoids, vincaalkaloids or derivatives thereof. In another embodiment, thechemotherapeutic agent is actinomycin-D, all-trans retinoic acidazacitidine, adriamycin azathioprine, bleomycin, camptothecin,carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide,cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin,epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil (5FU),gemcitabine, hydroxyurea, hydrogen peroxide, idarubicin, imatinib,mechlorethamine, mercaptopurine, methotrexate, mitomycin C,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,tioguanine, valrubicin, vinblastine, vincristine, vindesine, orvinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In one embodiment, themammal is a human. In another embodiment, the treating cancer in amammal comprises treating tumor hypoxia. In another embodiment, thetreating cancer in a mammal comprises inhibiting tumor angiogenesis. Inanother embodiment, the anti-nucleolin antibody or fragment thereof issubstantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin monoclonalantibody or fragment thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the toxin is diphtheria toxin, exotoxinA chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, phytolaca americanaprotein, pokeweed antiviral protein, momordica charantia inhibitor,curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,restrictocin, phenomycin, enomycin, calicheamicins or tricothecenestoxin. In another embodiment, the chemotherapeutic agent is analkylating agent, anthracycline, cytoskeletal disruptor, epothilone,inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleosideor nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

A method of treating or preventing cancer in a mammal comprisingadministering to said mammal a therapeutically effective amount of aanti-nucleolin antibody or fragment thereof, a toxin or chemotherapeuticagent and a pharmaceutically acceptable carrier, wherein said antibodyor fragment thereof specifically binds to a protein of SEQ ID No. 4. Inone embodiment, the mammal is a human. In another embodiment, thetreating cancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase 1, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of a anti-nucleolin antibody orfragment thereof and a pharmaceutically acceptable carrier, and furthertreating said mammal with radiation therapy, wherein said antibody orfragment thereof specifically binds to a protein of SEQ ID No. 4. In oneembodiment, the mammal is a human. In another embodiment, the treatingcancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (such as 10, 20, 30,40, 50, 60, 70, 80, 90, or 100%) of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 (such as 48,72, or 96) hours. In one embodiment, the autoimmune disease is alopeciagreata, ankylosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, asthma, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, diabetes mellitus (e.g., type 1),eosinophilic fascites, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis,Henoch-Schonlein purpura, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), IgA neuropathy,juvenile arthritis, lichen planus, lupus erthematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1 orimmune-mediated diabetes mellitus, myasthenia gravis, pemphigus-relateddisorders (e.g., pemphigus vulgaris), myelodysplastic syndrome,pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome,Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet'ssyndrome, Still's disease, lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In one embodiment, theautoimmune disease is alopecia greata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, asthma,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, diabetes mellitus (e.g., type 1), eosinophilicfascites, fibromyalgia-fibromyositis, glomerulonephritis, Graves'disease, Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonleinpurpura, idiopathic pulmonary fibrosis, idiopathic/autoimmunethrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erthematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, type 1 or immune-mediated diabetesmellitus, myasthenia gravis, pemphigus-related disorders (e.g.,pemphigus vulgaris), myelodysplastic syndrome, pernicious anemia,polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still'sdisease, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In one embodiment, theautoimmune disease is alopecia greata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, asthma,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, diabetes mellitus (e.g., type 1), eosinophilicfascites, fibromyalgia-fibromyositis, glomerulonephritis, Graves'disease, Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonleinpurpura, idiopathic pulmonary fibrosis, idiopathic/autoimmunethrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erthematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, type 1 or immune-mediated diabetesmellitus, myasthenia gravis, pemphigus-related disorders (e.g.,pemphigus vulgaris), myelodysplastic syndrome, pernicious anemia,polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still'sdisease, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (10, 20, 30, 40, 50,60, 70, 90, 100%) of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 (48, 72, or 96) hours. Inanother embodiment, the airway disease is asthma, chronic obstructivepulmonary disease, idiopathic pulmonary fibrosis, or inflammatorypneumonitis. In another embodiment, the mammal is a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof issubstantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In another embodiment,the airway disease is asthma, chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin antibody or fragment thereof is a monoclonal antibody orfragment thereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In another embodiment,the airway disease is asthma, chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin antibody or fragment thereof is a monoclonal antibody orfragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 20% of a population of MCF-7cells, when incubated with said MCF-7 cells and human AB serum for 48hours. In another embodiment, the virally infected cell is infected withan HIV virus. In another embodiment, the mammal is a human. In anotherembodiment, the anti-nucleolin monoclonal antibody or fragment thereofis substantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin antibody orfragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In another embodiment,the virally infected cell is infected with an HIV virus. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In another embodiment,the virally infected cell is infected with an HIV virus. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises an anti-nucleolin antibody orfragment thereof and said antibody or fragment thereof kills at least10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 (48, 72, or 96) hours. In one embodiment, thecondition or disease in a mammal characterized by increased surfaceexpression of nucleolin is macular degeneration, diabetic retinopathy,or inflammatory disease. In another embodiment, the mammal is a human.In another embodiment, the anti-nucleolin monoclonal antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises a human anti-nucleolin antibody orfragment thereof that specifically binds to a protein of SEQ ID No. 2.In one embodiment, the condition or disease in a mammal characterized byincreased surface expression of nucleolin is macular degeneration,diabetic retinopathy, or inflammatory disease. In another embodiment,the mammal is a human. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is substantially non-immunogenicto a human. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises an anti-nucleolin antibody orfragment thereof that specifically binds to a protein of SEQ ID No. 4.In one embodiment, the condition or disease in a mammal characterized byincreased surface expression of nucleolin is macular degeneration,diabetic retinopathy, or inflammatory disease. In another embodiment,the mammal is a human. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is substantially non-immunogenicto a human. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides an anti-nucleolin agent thatkills at least 50% of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 hours. In another embodiment,the said anti-nucleolin agent is substantially non-immunogenic to ahuman.

In another aspect the invention provides an anti-nucleolin agent thatkills more MCF-7 cells than MCF10A cells when incubated with separatepopulations of MCF-7 cells and MCF10A cells and heat inactivated serumfor 72 or 96 hours. In another embodiment, the said anti-nucleolin agentis substantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent thatkills more MCF-7 cells than MCF10A cells when incubated with separatepopulations of MCF-7 cells and MCF10A cells and human AB serum for 96hours. In another embodiment, the said anti-nucleolin agent issubstantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent thatspecifically binds to a protein of SEQ ID No. 4 and inihibits or killsone or more cancer cells that express nucleolin on their cell surface.In another embodiment, the said anti-nucleolin agent is substantiallynon-immunogenic to a human.

In another aspect the invention provides a method of determining alikelihood that a subject will develop cancer by detecting increasedcell surface nucleolin expression in one or more precancerous cells.

In another aspect the invention provides a antibody of any of theproceeding claims, wherein said antibody fragment is a Fab, Fab′,F(ab′).sub.2, or Fv fragment; diabodie; linear antibody; single-chainantibody; or a multispecific antibodt formed from an antibody fragment.

In another aspect the invention provides a method of any of theproceeding claims comprising the use of an antibody fragment, whereinsaid antibody fragment is a Fab, Fab′, F(ab′).sub.2, or Fv fragment;diabodie; linear antibody; single-chain antibody; or a multispecificantibodt formed from an antibody fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Over-expression of nucleolin and Bcl-2 proteins in CLL cells vs.normal B cells. Peripheral-blood lymphocytes were isolated from CLLpatients and healthy volunteers by density gradient centrifugation andthe B cells were purified by positive selection with magnetic-activatedcell separation (MACS) CD19 immunomagnetic beads. Nucleolin and Bcl-2protein levels were measured in S10 extracts of the cells by Westernblotting. The results were normalized to the values obtained from knownamounts of nucleolin and Bcl-2 protein external standards. The labels Nand C along the X-axis refer to normal B cells and CLL cells,respectively, from individual subjects. N=CD19+ B cells from healthyvolunteers; C=CD19+ CLL cells.

FIGS. 2A-B. Subcellular localization of nucleolin in CLL vs normal Bcells and MCF-7 vs MCF-10A cells. The intracellular localization ofnucleolin was determined by indirect immunofluorescence using mouseanti-human nucleolin mAb and a secondary FITC-conjugated anti-mouse IgG.Nuclei were counterstained with propidium iodide. FIG. 2A shows acomparison between CLL and normal B cells. FIG. 2B shows a comparisonbetween MCF-7 and MCF-10A cells.

FIG. 3. Subcellular localization of nucleolin in MCF-7 and MCF-10Acells. The intracellular localization of nucleolin was determined byindirect immunofluorescence using a monoclonal antibody against humannucleolin and a secondary FITC-conjugated anti-mouse IgG. Nuclei werecounterstained with propidium iodide. Cell morphology is shown as DICimages. Results are representatives of three separate experiments.

FIG. 4. Bone marrow biopsy from a patient with AML-M1 stained withanti-nucleolin antibody (fuchsin stain) shows intense nuclear staining.Interspersed non-leukemic marrow elements are either negative (dark bluecounterstain) or show only a nuclear blush. Magnification was 40×.

FIGS. 5A-B. Nucleolin overexpression in spleen sections from humanAML-engrafted mice and from human breast cancer cells. (FIG. 5A) Twowell-defined nodular human leukemic infiltrates present in the mousespleen. The leukemic infiltrates are positive for nucleolin (fuchsinimmunostain). Surrounding mouse spleen lymphocytes are negative fornucleolin immunostaining (blue counterstain). Magnification was 40×.(FIG. 5B) A tissue array with 50 cases of paraffin embedded formalinfixed human breast tumor and adjacent normal tissue (Biomax BR1006) wasstained with anti-nucleolin Mab MS3 (Santa Cruz 1:100) using Vectastainmouse kit, DAB substrate, and hematoxylin counterstain. Representativecases were imaged on an Olympus CKX41 at 100× magnification.

FIG. 6. Relative expression levels of bcl-2 hnRNA and bcl-2 mRNA in CLLand normal B cells. The levels of bcl2 hnRNA and bcl2 mRNA in CLL cellsfrom 4CLL patients and normal B cells from 4 healthy volunteers weredetermined by RT-PCR. Results are expressed as the means of 4determinations per group SEM. *P<0.001 compared with normal B cells.

FIG. 7. Decay of bcl-2 mRNA in extracts of CLL and normal B cells.5′-capped and polyadenylated [³²P]bcl-2-CR RNA and [³²]bcl-2-CR-ARE RNAwere incubated with S100 extracts prepared from either CLL cells from 4patients or normal B cells from 3 human volunteers. At the indicatedtimes aliquots of the reaction mixtures were removed and analyzed byPAGE and phosphorimaging. The results are expressed as the meanpercentage of full-length RNA remaining±SEM as a function of time.Symbols: -∘- CLL cell extract+[³²P]bcl-2- CR RNA; -●-, CLL cellextract+[³²P]bcl-2-CR-ARE RNA; -□-, normal B cell extract+[³²P]bcl-2-CRRNA; -≡-, normal B cell extract+[³²P]bcl-2-CR-ARE RNA; -▴-, normal cellextract+[³²P]bcl-2-CR-ARE RNA+280 nM purified recombinant nucleolin.

FIG. 8. Producing human mAbs in vitro. Tonsil B cells are isolated andefficiently immortalized with EBV, then induced to differentiate intoantibody-producing cells. Immortalized B cell libraries are plated intomultiple wells, then cell supernatants containing IgG are screened forbinding to recombinant nucleolin. Reactive cells are cloned by limitingdilution, expanded, and the IgG purified from the culture supernatant.Ig genes can be subcloned into producer cell lines for large scalerecombinant IgG production. See PCT/US2008/072124.

FIGS. 9A-B. Efficient EBV infection and induction of differentiation intonsil B cell libraries. (FIG. 9A) Flow cytometric analysis of tonsil Bcells 24 h post-infection with GFP-labeled EBV (EBfaV-GFP) compared withcontrol after conventional supernatant infection (top panels) or afterspinfection with concentrated virus (bottom panels). GFP fluorescenceindicates infection. (FIG. 9B) Four B95-8 EBV-immortalized tonsil B celllibraries were cultured with or without anti-IgM F(ab′)2, recombinantsoluble CD40L and Baff. Cell supernatants were assayed for IgM and IgGby ELISA after 5 (top panel) or 15 (bottom panel) days.

FIGS. 10A-B. Isolation of eight immortalized B cell lines producingnucleolin-specific human antibodies. (FIG. 10A) Culture supernatantsobtained from six human B cell lines producing anti-nucleolin antibodieswere screened by ELISA for binding to recombinant Δ1-283Nuc-(His)₆nucleolin (nucleolin lacking the N-terminal amino acids 1 through 283),using HRP-labeled goat anti-human IgG and colorimetric substrate fordetection. Binding was measured by spectrophotometry at 450 nm. Resultswere compared with that obtained from culture supernatants containinghuman anti-HA IgG or mouse anti-nucleolin MS3. (FIG. 10B) Eight B celllines producing anti-nucleolin antibodies were subcloned by limitingdilution cloning. Three weeks later, fewer than 10% of wells on each96-well plate contained small monoclonal colonies, which were thentested by ELISA for binding to recombinant nucleolin as described above.Nucleolin binding in the wells was assessed by measuring absorbance at450 nm.

FIG. 11. Isolation of human anti-nucleolin antibodies. Supernatants fromimmortalized human B cell libraries were screened by ELISA (top) andWestern blotting (bottom) for IgG binding to recombinantΔ1-283Nuc-(His)₆ nucleolin and/or endogenous nucleolin isolated fromMV411 leukemic cells.

FIG. 12. Binding of HuMAbs to cell surface nucleolin and cytotoxicity toMV4-11 leukemia cells. (FIG. 12A) Anti-nucleolin HuMabs or Rituxan^(R)(5 μg/ml) were incubated with MV4-11 or normal tonsil cells for 30 minon ice, washed, then incubated with APC-labeled anti-human IgG(Miltenyi). Flow cytometry was performed on a FACSCaliber, and medianfluorescence intensity (MFI) is shown.

FIG. 13. Complement-mediated killing of MV4-11 cells. Supernatants fromantibody-producing B cell lines were incubated with MV411 cells (50%v/v) for 16 h in the presence of human serum (25% v/v). Controls weretreated with staurosporine (25 μM) or media (untreated). Cells were thenstained with propidium iodide and annexin V-FITC, and analyzed by flowcytometry.

FIGS. 14A-C. Nucleolin specific killing of MCF-7 and MV4-11 AML cellsoccurs through complement dependent and independent mechanisms. (FIG.14A) MCF-7 cells were incubated with anti-nucleolin HuMAbs (2 μg/ml) inthe presence of human AB serum (+complement) or heat inactivated serum(−complement). Viability was determined by MTS assay at 48-96 h. (FIG.14B) MCF-7 and MCF10A cells were incubated with anti-nucleolin HuMAbs (2μg/ml, top panels; or 1 mg/ml, bottom panels) or AS1411 (20 μM), in thepresence of human AB serum (+complement) or heat inactivated serum(−complement). Viability was determined by MTS assay at 72 h (toppanels) and 96 h (bottom panels). (FIG. 14C) MV4-11 cells were incubatedwith anti-nucleolin HuMAbs (2 μg/ml) or AS1411 (20 μM), in the presenceof human AB serum (+complement) or heat inactivated serum (−complement).Viability was determined by MTS assay at 48-96 h.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides, human monoclonal antibodies that bindspecifically to human nucleolin, and methods of use thereof. Theseantibodies exhibit cytotoxicity towards cells expressing human nucleolinin the plasma membrane, such as cells involved in cancer, autoimmunedisorders, viral disorders. Therefore, the antibodies have therapeuticpotential for certain forms of cancer and autoimmune disorders and canalso be used as diagnostic agents.

I. NUCLEOLIN

A. General

Nucleolin is a multi-functional protein that binds to DNA, RNA and theexternal surface of the plasma membrane. The ability of nucleolin toperform numerous and diverse functions within the cell is related to themultiple structural domains within the protein. Its negatively chargedN-terminal domain regulates rDNA transcription by inducing nucleolarchromatin decondensation (Srivastava et al., 1989), while the centralglobular domain contains four RNA binding domains (RBDs) (Serin et al.,1997). It has been proposed that nucleolin, via binding of its RBD andits RGG-rich C-terminal domains to pre-ribosomal RNA, functions as anassembly factor by bringing together the correctly folded rRNA and othercomponents necessary for rRNA maturation and ribosome assembly (Ginistyet al., 2001). Nucleolin may also be involved in exporting ribosomes tothe cytoplasm while shuttling between the cytoplasm and nucleus(Srivastava and Pollard, 1999). The nucleolin gene coding and proteinsequences are listed in Table 1.

TABLE 1 Nucleolin Sequences SEQ ID NO: Identity Sequence 1 HumanCTTTCGCCTCAGTCTCGAGCTCTCGCTGGCCTTCGGG nucleolinTGTACGTGCTCCGGGATCTTCAGCACCCGCGGCCGCC codingATCGCCGTCGCTTGGCTTCTTCTGGACTCATCTGCGC sequenceCACTTGTCCGCTTCACACTCCGCCGCCATCATGGTGA AccessionAGCTCGCGAAGGCAGGTAAAAATCAAGGTGACCCCA numberAGAAAATGGCTCCTCCTCCAAAGGAGGTAGAAGAAG NM_005381ATAGTGAAGATGAGGAAATGTCAGAAGATGAAGAA XM_002342275GATGATAGCAGTGGAGAAGAGGTCGTCATACCTCAG AAGAAAGGCAAGAAGGCTGCTGCAACCTCAGCAAAGAAGGTGGTCGTTTCCCCAACAAAAAAGGTTGCAGTTGCCACACCAGCCAAGAAAGCAGCTGTCACTCCAGG CAAAAAGGCAGCAGCAACACCTGCCAAGAAGACAGTTACACCAGCCAAAGCAGTTACCACACCTGGCAAGAAGGGAGCCACACCAGGCAAAGCATTGGTAGCAACTCCTGGTAAGAAGGGTGCTGCCATCCCAGCCAAGGGGG CAAAGAATGGCAAGAATGCCAAGAAGGAAGACAGTGATGAAGAGGAGGATGATGACAGTGAGGAGGATGA GGAGGATGACGAGGACGAGGATGAGGATGAAGATGAAATTGAACCAGCAGCGATGAAAGCAGCAGCTGCTGCCCCTGCCTCAGAGGATGAGGACGATGAGGATGACG AAGATGATGAGGATGACGATGACGATGAGGAAGATGACTCTGAAGAAGAAGCTATGGAGACTACACCAGCCAAAGGAAAGAAAGCTGCAAAAGTTGTTCCTGTGAAA GCCAAGAACGTGGCTGAGGATGAAGATGAAGAAGAGGATGATGAGGACGAGGATGACGACGACGACGAAGATGATGAAGATGATGATGATGAAGATGATGACTGAGGAGGAAGAAGAGGAGGAGGAAGAGCCTGTCAAAGAA GCACCTGGAAAACGAAAGAAGGAAATGGCCAAACAGAAAGCAGCTCCTGAAGCCAAGAAACAGAAAGTGGAAGGCACAGAACCGACTACGGCTTTCAATCTCTTTGTTGGAAACCTAAACTTTAACAAATCTGCTCCTGAATTAAAAACTGGTATCAGCGATGTTTTTGCTAAAAATGATCTTGCTGTTGTGGATGTCAGAATTGGTATGACTAGGAAATTTGGTTATGTGGATTTTGAATCTGCTGAAGACCTGGAGAAAGCGTTGGAACTCACTGGTTTGAAAGTCTTT GGCAATGAAATTAAACTAGAGAAACCAAAAGGAAAAGACAGTAAGAAAGAGCGAGATGCGAGAACACTTTTGGCTAAAAATCTCCCTTACAAAGTCACTCAGGATGAATTGAAAGAAGTGTTTGAAGATGCTGCGGAGATCAGATTAGTCAGCAAGGATGGGAAAAGTAAAGGGATTGCTTATATTGAATTTAAGACAGAAGCTGATGCAGAGAAAACCTTTGAAGAAAAGCAGGGAACAGAGATCGATGGGCGATCTATTTCCCTGTACTATACTGGAGAGAAAGGTCAAAATCAAGACTATAGAGGTGGAAAGAATAGCACTTGGAGTGGTGAATCAAAAACTCTGGTTTTAAGCAACCTCTCCTACAGTGCAACAGAAGAAACTCTTCAGGAAGTATTTGAGAAAGCAACTTTTATCAAAGTACCCCAGAACCAAAATGGCAAATCTAAAGGGTATGCATTTATAGAGTTTGCTTCATTCGAAGACGCTAAAGAAGCTTTAAATTCCTGTAATAAAAGGGAAATTGAGGGCAGAGCAATCAGGCTGGAGTTGCAAGGACCCAGGGGATCACCTAATGCCAGAAGCCAGCCATCCAAAACTCTGTTTGTCAAAGGCCTGTCTGAGGATACCACTGAAGAGACATTAAAGGAGTCATTTGACGGCTCCGTTCGGGCAAGGATAGTTACTGACCGGGAAACTGGGTCCTCCAAAGGGTTTGGTTTTGTAGACTTCAACAGTGAGGAGGATGCCAAAGCTGCCAAGGAGGCCATGGAAGACGGTGAAATTGATGGAAATAAAGTTACCTTGGACTGGGCCAAACCTAAGGGTGAAGGTGGCTTCGGGGGTCGTGGTGGAGGCAGAGGCGGCTTTGGAGGACGAGGTGGTGGTAGAGGAGGCCGAGGAGGATTTGGTGGCAGAGGCCGGGGAGGCTTTG GAGGGCGAGGAGGCTTCCGAGGAGGCAGAGGAGGAGGAGGTGACCACAAGCCACAAGGAAAGAAGACGAAGTTTGAATAGCTTCTGTCCCTCTGCTTTCCCTTTTCCATTTGAAAGAAAGGACTCTGGGGTTTTTACTGTTACCTGATCAATGACAGAGGCTTCTGAGGACATTCCAAGACAGTATACAGTCCTGTGGTCTCCTTGGAAATCCGTCTAGTTAACATTTCAAGGGCAATACCGTGTTGGTTTTGACTGGATATTCATATAAACTTTTTAAAGAGTTGAGTGATAGAGCTAACCCTTATCTGTAAGTTTTGAATTTATATTGTTTCATCCCATGTACAAAACCATTTTTTTCCTACAAATAGTTTGGGTTTTGTTGTTGTTTCTTTTTTTTGTTTTGTTTTTGTTTTTTTTTTTTTTGCGTTCGTGGGGTTGTAAAAGAAAAGAAAGCAGAATGTITTATCATGGTITTTGCTTCAGCGGCTTTAGGACAAATTAAAAGTCAACTCTGG TGCCAGAAAAAAAAAAAAAAAA 2 HumanMVKLAKAGKNQGDPKKMAPPPKEVEEDSEDEEMSED nucleolinEEDDSSGEEVVIPQKKGKKAAATSAKKVVVSPTKKVA full lengthVATPAKKAAVTPGKKAAATPAKKTVTPAKAVTTPGKK amino acidGATPGKALVATPGKKGAAIPAKGAKNGKNAKKEDSDE sequenceEEDDDSEEDEEDDEDEDEDEDEIEPAAMKAAAAAPASE AccessionDEDDEDDEDDEDDDDDEEDDSEEEAMETTPAKGKKA numberAKVVPVKAKNVAEDEDEEEDDEDEDDDDDEDDEDDD NP_005372DEDDEEEEEEEEEEPVKEAPGKRKKEMAKQKAAPEAK XP_002342316KQKVEGTEPTTAFNLFVGNLNFNKSAPELKTGISDVFAKNDLAVVDVRIGMTRKFGYVDFESAEDLEKALELTGLKVFGNEIKLEKPKGKDSKKERDARTLLAKNLPYKVTQDELKEVFEDAAEIRLVSKDGKSKGIAYIEFKTEADAEKTFEEKQGTEIDGRSISLYYTGEKGQNQDYRGGKNSTWSGESKTLVLSNLSYSATEETLQEVFEKATFIKVPQNQNGKSKGYAFIEFASFEDAKEALNSCNKREIEGRAIRLELQGPRGSPNARSQPSKTLFVKGLSEDTTEETLKESFDGSVRARIVTDRETGSSKGFGFVDFNSEEDAKAAKEAMEDGEIDGNKVTLDWAKPKGEGGFGGRGGGRGGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGGDHKPQGKKTKF E 3 Humanaagaaggaaatggccaaacagaaagcagctcctgaagccaa nucleolingaaacagaaagtggaaggcacagaaccgactacggctttc codingaatctctttgttggaaacctaaactttaacaaatctgctc sequence forctgaattaaaaactggtatcagcgatgtttttgctaaaaa the deletiontgatcttgctgttgtggatgtcagaattggtatgactagg constructaaatttggttatgtggattttgaatctgctgaagacctgg amino acidagaaagcgttggaactcactggtttgaaagtctttggcaa sequencetgaaattaaactagagaaaccaaaaggaaaagacagtaag Δ1-283Nucaaagagcgagatgcgagaacacttttggctaaaaatctcc (lackingcttacaaagtcactcaggatgaattgaaagaagtgtttga residues 1-agatgctgcggagatcagattagtcagcaaggatgggaaa 283)agtaaagggattgcttatattgaatttaagacagaagctgatgcagagaaaacctttgaagaaaagcagggaacagagatcgatgggcgatctatttccctgtactatactggagagaaaggtcaaaatcaagactatagaggtggaaagaatagcacttggagtggtgaatcaaaaactctggttttaagcaacctctcctacagtgcaacagaagaaactcttcaggaagtatttgagaaagcaacttttatcaaagtaccccagaaccaaaatggcaaatctaaagggtatgcatttatagagtttgcttcattcgaagacgctaaagaagctttaaattcctgtaataaaagggaaattgagggcagagcaatcaggctggagttgcaaggacccaggggatcacctaatgccagaagccagccatccaaaactctgtttgtcaaaggcctgtctgaggataccactgaagagacattaaaggagtcatttgacggctccgttcgggcaaggatagttactgaccgggaaactgggtcctccaaagggtttggttttgtagacttcaacagtgaggaggatgccaaagctgccaaggaggccatggaagacggtgaaattgatggaaataaagttaccttggactgggccaaacctaagggtgaaggtggcttcgggggtcgtggtggaggcagaggcggctttggaggacgaggtggtggtagaggaggccgaggaggatttggtggcagaggccggggaggctttggagggcgaggaggcttccgaggaggcagaggaggaggaggtgaccacaagccacaaggaaagaagacg aagtttgaagtttaaac 4 HumanMAKQICAAPEAKKQKVEGTEPTTAFNLFVGNLNFNKSA nucleolinPELKTGISDVFAKNDLAVVDVRIGMTRKFGYVDFESAE deletionDLEKALELTGLKVFGNEIKLEKPKGKDSKKERDARTLL constructAKNLPYKVTQDELKEVFEDAAEIRLVSKDGKSKGIAYI amino acidEFKTEADAEKTFEEKQGTEIDGRSISLYYTGEKGQNQD sequenceYRGGKNSTWSGESKTLVLSNLSYSATEETLQEVFEKAT Δ1-283NucFIKVPQNQNGKSKGYAFIEFASFEDAKEALNSCNKREIE (lackingGRAIRLELQGPRGSPNARSQPSKTLFVKGLSEDTTEETL residues 1-KESFDGSVRARIVTDRETGSSKGFGFVDFNSEEDAKAA 283)KEAMEDGEIDGNKVTLDWAKPKGEGGFGGRGGGRGGFGGRGGGRGGRGGFGGRGRGGFGGRGGFRGGRGGGG DHKPQGKKTKFE Listed are the genomiccoding region and protein sequence of human nucleolin and N-terminaldeletion fragment of human nucleolin. Nucleolin is also known as C23,FLJ45706, F1159041, and NCL.

Human NCL gene consists of 14 exons with 13 introns and spansapproximately 11 kb. The nucleolin protein contains several functionaldomains that mediate its functions. The N-terminal part containsmultiple phosphorylation sites and is rich in acidic amino acids. Thecentral part of nucleolin includes four RNA binding domains (RBD) andthe C-terminal part contains glycine and arginine rich domain (termedRGG or GAR domain). (Farin et al., 2009)

In one embodiment, a region of nucleolin containing the RNA bindingdomain is used for generation of an isolated human anti-nucleolinantibody. In one embodiment, a region of nucleolin lacking residues1-283 (SEQ ID NO:4) is used for generation of an isolated humananti-nucleolin antibody. In one embodiment, the invention provides foran human anti-nucleolin antibody that specifically targets a RNAdomain-containing portion of nucleolin. In one embodiment, the inventionprovides for an isolated human anti-nucleolin monoclonal antibody thatspecifically targets a RNA domain-containing portion of nucleolin. Inone embodiment, the invention provides for an isolated humananti-nucleolin monoclonal antibody produced by a human B cell thatspecifically targets a RNA domain-containing portion of nucleolin. Inone embodiment, the interaction of a human anti-nucleolin antibody withnucleolin disrupts interactions of the nucleolin RNA domain with othermolecules. In one embodiment, the interaction of a human anti-nucleolinantibody with nucleolin disrupts interactions of the nucleolin RNAdomain with BCL-2.

A considerable body of evidence supports a role for nucleolin in mRNAstabilization. Nucleolin binds to the 3′-untranslated region (3′-UTR) ofamyloid precursor protein mRNA and stabilizes this mRNA (Westmark andMalter, 2001). It is also required for the stabilization of IL-2 mRNAthat occurs during T cell activation (Chen et al., 2000). More recentstudies have demonstrated that nucleolin binds to an A-U rich element(ARE) in the 3′-UTR of bcl-2 mRNA in HL-60 cells (Sengupta et al.,2004), chronic lymphocytic leukemia (CLL) cells (Otake et al., 2007),and MCF-7 breast cancer cells (Soundararajan et al., 2008). Binding ofnucleolin to the bcl-2 ARE stabilizes bcl-2 mRNA by protecting it fromribonuclease degradation, while shRNA knockdown of nucleolin in MCF-7cells leads to bcl-2 mRNA instability and decreased levels of bcl-2protein (Soundararajan et al., 2008).

Nucleolin is present on the external surface of various types of tumorcells Otake et al., 2007; Soundararajan et al., 2008; Chen et al., 2008;Hovanessian et al., 2000; Sinclair and O'Brien, 2002), despite its lackof a transmembrane domain or signal sequence (Srivastava et al., 1989;Lapeyre et al., 1987). Results show that nucleolin is not secreted fromeither MV4-11 cells or K-562 cells into the tissue culture medium(Soundararajan et al., 2009). This suggests that the presence ofnucleolin on the cell surface is not the result of adsorption ofsecreted nucleolin by macromolecules on the cell surface of tumor cells.However, nucleolin undergoes extensive posttranslational modification(Srivastava et al., 1989; Lapeyre et al., 1987). It has been isolated asa glyco-phospho-protein from the surface of various types ofproliferating cells (Hovanessian et al., 2000; Pfeifle and Anderer,1983). It is also possible that palmitoylation, prenylation, ormyristoylation of nucleolin may allow for insertion or anchoring ofthese hydrophobic regions of the protein into the plasma membrane. It isthought that nucleolin functions as a shuttling protein between theplasma membrane and nucleus (Hovanessian et al., 2000). In proliferatingtumor cells, nucleolin is often associated with endocytotic vesiclesthat invaginate from the plasma membrane (Hovanessian et al., 2000).Nucleolin also acts as a cell surface receptor for various ligands,since ligands bound to nucleolin within these vesicles becomeinternalized in a temperature-dependent process. For example, plasmamembrane nucleolin has been reported to function as a receptor forintimin-γ of E. coli (Sinclair and O'Brien, 2002), the anti-HIV agentmidkine (Said et al., 2002), laminin-1 (Kibbey et al., 1995), DNAnanoparticles (Chen et al., 2008), and the anti-angiogenic pseudopeptideHB-19 (Destouches et al., 2008). Nucleolin is an important protein inthe nucleolus involved in ribosome biogenesis and maturation inexponentially growing eukaryotic cells. In this regard, one importantfunction of nucleolin is as a shuttling protein between cytoplasm andnucleus involving RNA processing and other cell biological process.While in normal cellular physiology, nucleolin is localizedpredominantly in the nucleolus and cytoplasm, under certain conditions,especially in various disease states it has also been shown to bepresent in a phosphorylated form on the cell surface. In this regard,nucleolin in the cell membrane serves as a binding protein for a varietyof ligands that drive cell proliferation, differentiation, adhesion,mitogenesis and angiogenesis.

B. Nucleolin in Cancer

Several lines of evidence suggest that nucleolin is an excellent tumorantigen for antibody-based immunotherapy. Nucleolin is overexpressed inthe plasma membrane and cytoplasm a variety of human tumors includinghuman chronic lymphocytic leukemia (CLL) (Otake et al., 2007), acutemyeloid leukemia (AML) (Soundararajan et al., 2008), and breast cancercells (Soundararajan et al., 2008), but not in normal CD19+ B cells(Otake et al., 2007), CD33+ myeloid cells (Gattoni-Celli et al., 2009),nor in normal mammary epithelial cells (Soundararajan et al., 2008). Itis of interest that AML blast cells from patients that engraft inNOD/SCID mice show intense nucleolin staining in the plasma membrane andcytoplasm while the normal mouse bone marrow cells and spleenlymphocytes were negative for nucleolin (Gattoni-Celli et al., 2009).

The nucleolin targeting aptamer, AS1411, targets human nucleolin. Plasmamembrane nucleolin was recently reported to be a receptor for AS1411 inhuman MV4-11 leukemia cells (Soundararajan et al., 2009).

AS1411 binds to nucleolin that is overexpressed on the external surfaceof tumor cells and gains intracellular access when nucleolin is shuttledfrom the plasma membrane to the cytoplasm and nucleus. AS1411 has beenshown to exhibit antiproliferative activity in a broad set of cancercell lines that over-express nucleolin (Table 2).

TABLE 2 Cancer Cell Lines That Over-express Nucleolin and/or are KilledSubsequent to Nucleolin Inhibition Cancer Type: Cell Line: Lung cancerA549, NCI-H322M, NCI-H460, EKVX, HOP-92, NCI- H299, CaLu1, NCI-H1385,NCI-H82, CaLu6 Breast cancer MCF7, T-47D, BT-549, MDA-N, MDA-MB-231,ZR7S-1 Prostate cancer DU145, PC-3, CA-HPV-10 Colon cancer HCC 2998,HT-29, KM12, HCT-116, SW620, HCT-15, LS174T Pancreatic PANC-1,MIA-PaCa-2, PANC-1 cancer Renal cell 786-0, CAKI-1, RXF393, TK10, A498,ACHN, SN12C carcinoma Ovarian cancer IGROV, OVCAR-3, OVCAR-4, OVCAR-5Cervical cancer HeLa Leukemia & CCRF-CEM, SR, HL60, K-562, RPMI-6226,U937, Lymphoma Meg0, MV4-11 Melanoma LOX-IMVI, SK-MEL-2, A375,SK-MEL-28, MDA-MB- 435 Glioblastoma SF-268, U87-MG Neuroblastoma IMR 32,Lan 5 Sarcoma HT-1080 Gastric cancer KATOIII, HGC27 Data from NCI TumorCell Line Screen of AS1411 (≧50% growth inhibition at 6.3 μM). (Bates etal., 2009).Anti-nucleolin antibodies can also exploit the shuttling function ofplasma membrane nucleolin and become internalized after binding to cellsurface nucleolin. Of significance in the present application is thefinding that the incubation of human tumor vascular endothelial cells,grown in nude mice or matrigel plugs, with a polyclonal anti-nucleolinantibody resulted in downregulation of bcl-2 mRNA levels and inductionof apoptosis (Fogal et al., 2009). This suggests that anti-nucleolinantibodies can elicit anti-tumor effects through intracellularmechanisms, and/or to antibody-dependent cellular cytotoxicity (ADCC)and complement-dependent cellular cytotoxicity (CDCC).

C. Antibodies or Fragments Thereof

In one embodiment any of the methods disclosed herein can be practicedwith an anti-nucleolin antibody or fragment thereof. In one embodimentan anti-nucleolin antibody or fragment thereof is used to detect a cellexpressing nucleolin on its surface. In another embodiment ananti-nucleolin antibody or fragment thereof is used to inhibit or kill acell expressing nucleolin on its surface. In another embodiment ananti-nucleolin antibody or fragment thereof is used to treat or preventa neoplastic disease (e.g. cancer), an autoimmune disease, aninflammatory disease or condition, a respiratory disease, a viralinfection, or macular degeneration.

In one embodiment an anti-nucleolin antibody or fragment thereof isconjugated, linked or fused to a toxin, chemotherapeutic, animmunostimulatory nucleic acid sequence (e.g., a CpG sequence), aradionuclide or an immunotherapeutic. In another embodiment ananti-nucleolin antibody or fragment thereof is conjugated, linked orfused to a radionuclide, a fluorophore, a chemilluminescent compound, afluorescent compound, or an enzyme. In another embodiment anti-nucleolinantibody or fragment thereof is used to contact a cell expressingnucleolin on its surface. In one embodiment the cell is pre-cancerouscell, a cancer cell or an immune cell.

In one embodiment the anti-nucleolin antibody fragment thereof is ahuman anti-nucleolin antibody or fragment. In one embodiment theanti-nucleolin antibody fragment thereof is a non-human anti-nucleolinantibody fragment thereof. In one embodiment the anti-nucleolin antibodyfragment thereof is a chimeric anti-nucleolin antibody fragment thereof.In one embodiment the anti-nucleolin antibody fragment thereof is ahumanized anti-nucleolin antibody fragment thereof.

In one embodiment an anti-nucleolin antibody fragment thereof isgenerated from an anti-nucleolin antibody. In one embodiment theanti-nucleolin antibody fragment has the same binding specificity tohuman nucleolin as the parent antibody. In another embodiment theanti-nucleolin antibody fragment has improved binding specificity tohuman nucleolin as the parent antibody. In one embodiment theanti-nucleolin antibody fragment has the same binding affinity to humannucleolin as the parent antibody. In another embodiment theanti-nucleolin antibody fragment has improved affinity to humannucleolin as the parent antibody. In one embodiment an anti-nucleolinantibody or fragment thereof is a human anti-nucleolin antibodyfragment.

Antibody fragments” comprise a portion of an intact antibody, preferablycomprising the antigen binding region thereof. Examples of antibodyfragments include Fab, Fab′, F(ab′).sub.2, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′).sub.2 fragmentthat has two antigen-combining sites and is still capable ofcross-linking antigen.

“Fv” is a minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′).sub.2 antibody fragments originally were produced as pairsof Fab′ fragments which have hinge cysteines between them. Otherchemical couplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., PNAS USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med. 9:129-134(2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations can be advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by a variety of techniques, including, for example, the hybridomamethod (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo etal., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,PNAS USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.Methods 284(1-2): 119-132 (2004), and technologies for producing humanor human-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., PNAS USA 90: 2551 (1993); Jakobovits et al., Nature362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993);U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;and 5,661,016; Marks et al, Bio/Technology 10: 779-783 (1992); Lonberget al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813(1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996);Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13: 65-93 (1995).

The modifier “polyclonal” indicates the character of the antibody asbeing obtained from a source of a nonhomogeneous population ofantibodies. A polyclonal antibody comprises more than one antibody, suchas 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antibodies.

The monoclonal antibodies herein include human, non-human, humanized and“chimeric” antibodies. “Chimeric” antibodies in which a portion of theheavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; and Morrison et al., PNAS USA 81:6851-6855(1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., PNAS USA,103:3557-3562 (2006) regarding human antibodies generated via a humanB-cell hybridoma technology.

II. PREPARING HUMAN MONOCLONAL ANTIBODIES FROM IGM⁺ B-CELLS

The following are descriptions of the general procedures for obtaining ahuman monoclonal antibody against nucleolin. These procedures areexemplary and can be modified while retaining the essential aspects ofthe invention.

A. Obtaining IgM⁺ B-Cell Populations

To prepare B-cells from tonsils, tonsil tissue is mixed with anantibiotic, chopped and minced to approximately 1 mm³ pieces, followedby gentle grinding of tonsil pieces and straining through a nylonstrainer. The suspension is then centrifuged on a Ficoll cushion. Theboundary layer containing mononuclear cells is extracted, washed andre-suspended in DPBS. Further enrichment (>95%) can be achieved bynegative selection using antibodies and magnetic beads

Non-human mammalian, chimeric, polyclonal (e.g., sera) and/or monoclonalantibodies (Mabs) and fragments (e.g., proteolytic digestion or fusionprotein products thereof) are potential therapeutic agents are oftenused in an attempt to treat certain diseases. However, such antibodiesor fragments can elicit an immune response when administered to humans.Such an immune response can result in an immune complex-mediatedclearance of the antibodies or fragments from the circulation, and makerepeated administration unsuitable for therapy, thereby reducing thetherapeutic benefit to the patient and limiting the readministration ofthe antibody or fragment. For example, repeated administration ofantibodies or fragments comprising non-human portions can lead to serumsickness and/or anaphalaxis. In order to avoid these and other problems,a number of approaches have been taken to reduce the immunogenicity ofsuch antibodies and portions thereof, including chimerization andhumanization. These and other approaches, however, still can result inantibodies or fragments having some immunogenicity, low affinity, lowavidity, or with problems in cell culture, scale up, production, and/orlow yields. Thus, such antibodies or fragments can be less than ideallysuited for manufacture or use as therapeutic proteins. Thus, an antibodyexpressed from human cells can avoid many of these issues and besubstantially non-immunogenic to a human. “Immunogenic” as used hereinrefers to possessing the ability to elicit an immune response.

In one embodiment, a human anti-nucleolin antibody expressed by a humanB cell is substantially non-immunogenic a human. In one embodiment, anisolated human anti-nucleolin monoclonal antibody expressed by a human Bcell is substantially non-immunogenic a human.

To prepare B-cells from peripheral blood, venous blood is drawn intosyringes containing heparin sodium to prevent coagulation, diluted, andcentrifuged on a Ficoll cushion. The boundary layer containingmononuclear cells is extracted, washed and re-suspended in DPBS. Furtherenrichment can be achieved as stated above.

B. EBV Immortalization

For infection by inoculation with EBV supernatant, B-cells areresuspended at 10⁶ to 10⁷ cells per ml in complete RPMI media, and mixedwith an equal volume of filtered EBV supernatant, then incubated for 4hours at 37° C. and 5% CO₂. The culture volume can be adjusted by theaddition of complete RPMI media, such that infected cells wereresuspended for cell culture at a desired concentration (generally 10⁵to 10⁶ cells per ml). Cells are then dispensed into multi-well platesand transferred to a tissue culture incubator at 37° C. and 5% CO₂.

For spin-fection, B-cells are resuspended at 10⁶ to 10⁷ cells per ml incomplete RPMI media, and mixed with an equal volume of 10-foldultrafiltration concentrated EBV and placed in a well of a 6-well tissueculture plate. The plate is then centrifuged at 900 g for 1 hr atambient temperature, at which time infected cells are re-suspended incomplete RPMI media at a desired concentration (generally 10⁵ to 10⁶cells per ml), dispensed into multi-well plates and transferred to atissue culture incubator at 37° C. and 5% CO₂.

Optionally, B-cells may be contacted with Toll Like Receptor (TLR)ligands at the time of or subsequent to the infection. The ligands maybe added at the following final concentrations: Pam3CSK4 (0.1-1 μg/ml),Zymosan (1-10 μg/ml), poly I:C (1-25 μg/ml), LPS (1-5 μg/ml), Imiquimod(1 μg/ml), and/or CpG (1-10 μg/ml).

Infectivity varies based upon route of infection. Infection of tonsil Bcells by inoculation with EBV supernatant results in immortalization ofapproximately 1-5% of B cells. Addition of TLR ligands approximatelydoubles infection efficiency. Infection of tonsil B cells byspin-fection with concentrated virus increases infection efficiency tovirtually 100% after 24 hours.

C. Culturing to Induce Immunoglobulin Isotype Class Switching and IgGSecretion

To induce B-cell differentiation, immunoglobulin isotype classswitching, and/or IgG secretion, cytokines and other signaling agentsare added to EBV infected B-cells immediately after infection, 16 to 20hr after infection, and/or sequentially at weekly intervals (2, 3, 4 or5 times). Agents may be diluted in media and added to cells at thefollowing final concentrations: recombinant human interleukins (IL)IL-4, 0.2 ng/ml; IL-5, 0.2 ng/ml; IL-6, 0.1 ng/ml; IL-9, 0.2 ng/ml;IL-10, 0.24 ng/ml; IL-13, 1 ng/ml; recombinant human interferon-α2a(IFN-α2a), 2,000 IU/ml; recombinant human BAFF, 1 ng/ml; recombinanthuman soluble CD40L, 5 ng/ml; goat anti-human, IgM F(ab′)₂, 1.4 μg/ml(amounts are approximate). Particular combinations comprise anti-IgMF(ab′)₂, CD40L+/−BAFF; anti-IgM F(ab′)₂, IL-6+/−BAFF; anti-IgM F(ab′)₂,CD40L, IL-6+/−BAFF; anti-IgM F(ab′)₂, CD40L, IL-6+/−IL4; and anti-IgMF(ab′)₂, CD40L, and IL-9+/−IL-13.

The initiation of immunoglobulin isotype class switching can be detectedas early as 5 days following exposure to the cytokine/growthfactor/signaling agent cocktail, and the process continues for thefollowing 10 days.

D. Selection of Immortalized B-Cells

Following exposure to the cytokine/growth factor/signaling agentcocktail, culture supernatants are collected about once a week or atdays 10-20 post-infection from immortalized tonsil and blood B-cellcultures, pooled, and tested using an ELISA or other screening format,such as dot blot, or flow cytometry, Western blotting, orinhibition/promotion of functional activity. Antigen may be bounddirectly or through capture antibodies onto the wells of a polystyrene(e.g., 96-well) plate and allowed to bind, e.g., overnight. Plates arethen washed, blocked, and contacted with immortalized B cell culturesupernatant samples or controls in triplicate or other replicates.Subsequently, the plate is washed extensively, and then e.g., alkalinephosphatase (AP)-coupled goat anti-human IgG or other labeled secondaryantibody is added for detection of bound IgG by e.g. AP conversion ofcolorimetric substrate p-nitrophenyl phosphate disodium salt.

Based upon the discussion above, immunoglobulin isotype class switchingand/or IgG secretion starts immediately after and can be detected asearly as 5 days following exposure to the cytokine/growthfactor/signaling agent cocktail. IgG levels increase in the supernatantover the next 10 days. Thus, from about 7-21 days, about 10-21 days,about 7-10 days or about 10-14 days, or at 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or 21 days, one will select B-cells that haveundergone immunoglobulin isotype class switching and secrete IgG.

III. CLONING AND EXPRESSION OF HUMAN Ig LIGHT AND HEAVY CHAINS

Various methods can be employed for the cloning and expression of humanimmunoglobulin light and heavy chain sequences. Weltschof et al. (1995),incorporated herein by reference, describes in detail the methods usedby the inventors. The variable regions, or variable+constant regions,can be cloned.

Other techniques, such as those described by Takekoshi et al. (2001),are also useful. In that reference, total cellular RNA was isolated frompelleted cells using a commercial kit (RNeasy mini kit, Qiagen). Usingrandom 9-mers, nucleotides and reverse transcriptase (Takara, RNA-PCRkit, Ohtsu), cDNAs were synthesized and were amplified by the polymerasechain reaction (PCR), with heavy and light chain primers specific forhuman immunoglobulins (Ig). A “touchdown” PCR protocol was employed,i.e., three cycles each of denaturation at 95° C. for 1 min, annealingfor 1 min, and elongation at 72° C. for 2 min, for a total of 11 cycles.The annealing temperature was varied from 65-55° C. in steps of 1° C.The touchdown cycles were followed by 25 cycles using an annealingtemperature of 55° C. The resultant PCR product was gel-purified inagarose and extracted using Qiaquick spin-columns (Qiagen). The lightchain and heavy chain Fc genes were then cloned into the NheI/AscI andthe SfiI/NotI sites of the expression vector pFab1-His2. The ligatedpFab1-His2 vectors with the light chain (κ and λ) and Fc heavy chaingenes (γ and μ) were introduced into competent E. coli JM109 cells(Toyobo, Osaka). After transformation, the E. coli cells were platedonto Luria-Bertani (LB)/ampicillin (50 μg/ml) plates. Isolated bacterialcolonies were incubated at 30° C. in 2 ml of Super Broth (SB) withampicillin (50 μg/ml) and MgCl₂ (1.5 mM).Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce productionof the Fab protein. Cells from the bacterial cultures were pelleted,resuspended in 0.3 ml of B-PER (Pierce) with a protease inhibitorcocktail (Complete, Boehringer Mannheim), and shaken for 5 min at roomtemperature. Cell lysates were centrifuged at 15,000 G for 10 min, andthe resultant supernatant containing the Fab antibody portion wascollected.

The foregoing is purely exemplary and other methods can be employed.

IV. ANTIBODY PRODUCTION

Once cloned, the nucleic acids for the human light and heavy chains canbe inserted into appropriate expression vectors and transferred intohost cells (e.g., antibody-producing cells) that support production ofantibodies. Particular cell lines contemplated for production are 293cells, CHO cells, COS cells or various forms of myeloma cells, somelacking IgG. These cells can be exploited for human MAb production intwo basic ways. First, myelomas or immortalized cells can be injected(often into the peritoneal cavity) into a histocompatible animal of thetype that was used to provide the somatic and myeloma cells for theoriginal fusion (e.g., a syngeneic mouse), or into an immunodeficientanimal for injection of noncompatible cells. Optionally, the animals areprimed with a hydrocarbon, especially oils such as pristane(tetramethylpcntadecane) prior to injection. The injected animaldevelops tumors secreting the specific monoclonal antibody produced bythe transfected myeloma. The body fluids of the animal, such as serum orascites fluid, can then be tapped to provide human MAbs in highconcentration. Second, the individual cell lines could be cultured invitro, where the human MAbs are naturally secreted into the culturemedium from which they can be readily obtained in high concentrations.

Human MAbs produced by either means can be further purified, if desired,using filtration, centrifugation and various chromatographic methodssuch as HPLC or affinity chromatography. Fragments of the monoclonalantibodies of the invention can be obtained from the monoclonalantibodies so produced by methods which include digestion with enzymes,such as pepsin or papain, and/or by cleavage of disulfide bonds bychemical reduction.

In one embodiment a human anti-nucleolin antibody is produced from animmortalized human B cell. In one embodiment a human anti-nucleolinantibody is produced using a method such as one set forth inPCT/US2008/072124 or U.S. patent application Ser. No. 12/671,936, whichare herein incorporated by reference in their entirety.

In one embodiment, the cDNA of an isolated human anti-nucleolin antibodymay be produced by cloning cDNA or genomic DNA encoding theimmunoglobulin light and heavy chains of the anti-nucleolin antibodyfrom a hybridoma cell (by fusing a specific antibody-producing B cellwith a myeloma) that produces an antibody homolog according to thisinvention. In one embodiment, an isolated human anti-nucleoline antibodyis produced by a human B cell. In one embodiment, a cell is transfectedby one or more polynucleotide sequences isolated from a human B cellwhere the polynucleotide sequence encodes for human anti-nucleolinantibody. The cDNA or genomic DNA encoding the polypeptides can beinserted into expression vectors so that both genes are operativelylinked to their own transcriptional and translational expression controlsequences. The expression vector and expression control sequences canthen be chosen to be compatible with the expression host cell used.Typically, both genes are inserted into the same expression vector.

Prokaryotic or eukaryotic cells can be used as expression hosts.Expression in eukaryotic host cells is preferred because such cells aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active antibody. However, any antibodyproduced that is inactive due to improper folding may be renaturableaccording to well known methods (Kim and Baldwin, 1982). It is possiblethat the host cells will produce portions of intact antibodies, such aslight chain dimers or heavy chain dimers, which also are antibodyhomologs according to the present invention.

It will be understood that variations on the above procedure are withinthe scope of the present invention. In one embodiment, a host cell istransformed with DNA encoding either the light chain or the heavy chain(but not both) of an antibody homolog of this invention. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary fornucleolin binding. The molecules expressed from such truncated DNAmolecules are antibody homologs according to this invention. In oneembodiment, bifunctional antibodies are produced in which one heavy andone light chain are homologs of a human anti-nucleolin antibody and theother heavy and light chain are specific for an antigen other thannucleolin, or another epitope of nucleolin.

In one embodiment, DNA encoding an isolated human anti-nucleolinantibody is transferred to a preferred mammalian cell line forexpression in “production” or commercial amounts. It has long beenrecognized that Chinese Hamster Ovary cells (CHO cells) make excellentexpression vehicles for recombinant or non-endogenous DNA. See U.S. Pat.No. 4,816,567. There has been developed a series of DHFR deficient CHOcell strains, which permit the amplification of inserted DNA encodingspecific proteins or DNA sequences, as set forth in U.S. Pat. No.5,981,214. Examples of additional mammalian cell lines for expression in“production” or commercial amounts include, but are not limited to293HEK cells, HeLa cells, COS cells, NIH3T3 cells, Jurkat Cells., NSOcells and HUVEC cells. Other mammalian cell lines suitable for theexpression of recombinant proteins have been identified in theliterature, and can be equally suitable for use in the invention of thisapplication.

A. Amino Acid Sequence Variants of Antibodies

In one embodiment, the anti-nucleolin antibody of the inventioncomprises a modified amino acid sequence compared to wild type. Forexample, it can be desirable to improve the binding affinity and/orother biological properties of the antibody. Amino acid sequencevariants of the anti-nucleolin antibodies are prepared by introducingappropriate nucleotide changes into the nucleic acid encoding theanti-nucleolin antibody chains, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the anti-nucleolin antibody. Any combination of deletion, insertion,and substitution is made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid changes also may alter post-translational processes of theanti-nucleolin antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theanti-nucleolin antibody that are preferred locations for mutagenesis iscalled “alanine scanning mutagenesis,” as described by Cunningham andWells Science (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and gluand replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with the antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed anti-nucleolinantibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean anti-nucleolin antibody with an N-terminal methionyl residue or theantibody fused to an epitope tag. Other insertional variants of theanti-nucleolin antibody molecule include the fusion to the N- orC-terminus of the antibody of an enzyme or a polypeptide which increasesthe serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the anti-nucleolinantibody molecule removed and a different residue inserted in its place.Substantial modifications in the biological properties of theanti-nucleolin antibody are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu,ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4)basic: asn, gln, his, lys, arg; (5) residues that influence chainorientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions may also be done and entail exchanging amember of one of these classes for another class. Any cysteine residuenot involved in maintaining the proper conformation of the antagonistalso may be substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the antagonist to improve its stability(particularly where the anti-nucleolin antibody is an antibody fragmentsuch as an Fv fragment).

A type of substitution variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitution variants is affinity maturation using phagedisplay. Briefly, several hypervariable region sites (e.g. 6-7 sites)are mutated to generate all possible amino substitutions at each site.The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g.,antagonist activity) as herein disclosed. In order to identify candidatehypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, or inaddition, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand nucleolin. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies with superiorproperties in one or more relevant assays may be selected for furtherdevelopment.

B. Glycosylation Variants of Antibodies

In one embodiment, the anti-nucleolin antibody is modified to havealtered glycosylation compared to wild type antibody. Antibodies areglycosylated at conserved positions in their constant regions (Jefferisand Lund, 1997; Wright and Morrison, 1997). The oligosaccharide sidechains of the immunoglobulins affect the protein's function (Boyd etal., 1996; Wittwe and Howard, 1990), and the intramolecular interactionbetween portions of the glycoprotein which can affect the conformationand presented three-dimensional surface of the glycoprotein (Jefferisand Lund, 1996). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. For example, it has been reported that in agalactosylatedIgG, the oligosaccharide moiety ‘flips’ out of the inter-CH2 space andterminal N-acetylglucosamine residues become available to bind mannosebinding protein (Malhotra et al., 1995). Removal by glycopeptidase ofthe oligosaccharides from CAMPATH-1H (a recombinant humanized murinemonoclonal IgG1 antibody which recognizes the CDw52 antigen of humanlymphocytes) produced in Chinese Hamster Ovary (CHO) cells resulted in acomplete reduction in complement mediated lysis (CMCL) (Boyd et al.,1996), while selective removal of sialic acid residues usingneuraminidase resulted in no loss of DMCL. Glycosylation of antibodieshas also been reported to affect antibody-dependent cellularcytotoxicity (ADCC). In particular, CHO cells withtetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al. 1999).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. Nucleic acid molecules encoding amino acidsequence variants of the anti-nucleolin antibody are prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of the anti-nucleolin antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected (see, e.g., Hseet al., 1997). In addition to the choice of host cells, factors whichaffect glycosylation during recombinant production of antibodies includegrowth mode, media formulation, culture density, oxygenation, pH,purification schemes and the like. Various methods have been proposed toalter the glycosylation pattern achieved in a particular host organismincluding introducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H). In addition, the recombinant host cell canbe genetically engineered, e.g. make defective in processing certaintypes of polysaccharides. These and similar techniques are well known inthe art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

C. Other Modifications of Antibodies

In one embodiment, an anti-nucleolin antibody is formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al. (1985);Hwang et al. (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. (1982) via adisulfide interchange reaction. A chemotherapeutic agent (such asDoxorubicin) is optionally contained within the liposome. See Gabizon etal. (1989).

In one embodiment, an anti-nucleolin antibody is used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81/01145) to anactive drug. See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active form exhibiting the desired biologicalproperties.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as “abzymes,” can be used to convert the prodrugs ofthe invention into free active drugs (see, e.g., Massey, 1987).Antibody-abzyme conjugates can be prepared as described herein fordelivery of the abzyme to a desired cell population.

The enzymes can be covalently bound to the anti-nucleolin antibodies bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (see, e.g., Neubergeret al., 1984).

In one embodiment of the invention, an anti-nucleolin antibody comprisesan antibody fragment, rather than an intact antibody. In this case, theantibody fragment may be modified in order to increase its serumhalf-life. This may be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis). See WO 96/32478 published Oct. 17, 1996.

The salvage receptor binding epitope generally constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fe region (e.g., of an IgG)and transferred to the CH1, CH3, or V_(H) region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the antibody fragment.

In one embodiment, an anti-nucleolin antibody is modified by covalentlinkages. Covalent linkages may include but are not limited to bychemical synthesis or by enzymatic or chemical cleavage of the antibody.Other types of covalent modifications of the antibody are introducedinto the molecule by reacting targeted amino acid residues of theantibody with an organic derivatizing agent that is capable of reactingwith selected side chains or the N- or C-terminal residues. Exemplarycovalent modifications of polypeptides are described in U.S. Pat. No.5,534,615, specifically incorporated herein by reference. One type ofcovalent modification of the antibody comprises linking the antibody toone of a variety of nonproteinaceous polymers, e.g., polyethyleneglycol, polypropylene glycol, or polyoxyalkylenes, in the manner setforth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337.

In another embodiment, an anti-nucleolin antibody (such as a humanantibody) is modified by fusing, or conjugating it to another,heterologous polypeptide or amino acid sequence.

In one embodiment, an human anti-nucleolin antibody is modified tocomprise targeted immunoconjugate moieties which enable the effectivegeneration of innate and adaptive immune responses against tumors orpathogens. In one embodiment, an isolated human anti-nucleolinmonoclonal antibody is modified to comprise targeted immunoconjugatemoieties which enable the effective generation of innate and adaptiveimmune responses against tumors or pathogens. In one embodiment, anisolated human anti-nucleolin monoclonal antibody produced by a human Bcell is modified to comprise targeted immunoconjugate moieties whichenable the effective generation of innate and adaptive immune responsesagainst tumors or pathogens. The immunoconjugates can be capable ofsimultaneously satisfying multiple key requirements for mountingeffective antibody- and/or cell-mediated immune responses against thetargeted tumor or pathogen, which include but are not limited to: (i)Inducing or augmenting uptake and cross-presentation of tumor- orpathogen antigen(s) or antigenic determinant(s) by antigen presentingcells (APC)/dendritic cells (DC); (ii) promoting the maturation ofdendritic cells (DCs) in the target cell milieu; (iii) providing CD4+ Tcell help to generate CD8+ T cell memory and antibodies against thetumor or pathogen; (iv) sensitizing the targeted tumor cell to antibodydependent cell cytotoxicity (ADCC) and T-cell mediated death. Suchimmunoconjugated antibodies can be used for targeted immunotherapy orimmunoprophylaxis of neoplastic diseases, infectious diseases, and otherdisorders. For example, pattern recognition receptors (PRRs), such asToll Like Receptors, recognize pathogen-associated molecular patterns(PAMPs) expressed by diverse infectious microorganisms (bacteria, fungi,protozoa, viruses) and molecules released by damaged host tissues(damage associated molecular patterns/alarmins). The addition of a PAMPconjugated to a isolated human anti-nucleolin antibody provides a moietycomprising a nucleic acid or protein that is recognized by a PRR,ultimately leading to an immune response which eliminates the targetcell with the anti-nucleolin antibody bound to it. Examples of PAMPSthat can be conjugated to an anti-nucleolin antibody include but arelimited to known viral and pathogenic epitopes, such aspolyinosine-polycytidylic acid, lipopolysaccharide (LPS), lipid A,flagellin, GU-rich short single-stranded RNA, unmethylatedCpG-oligodeoxynucleotides.

In one embodiment an anti-nucleolin antibody (such as a human antibody)is fused or conjugated with a tag polypeptide which provides an epitopeto which an anti-tag antibody can selectively bind. The epitope tag canbe placed at the amino- or carboxyl-terminus of the anti-nucleolinantibody. The presence of such epitope-tagged forms of an anti-nucleolinantibody can be detected using an antibody against the tag polypeptide.Also, provision of the epitope tag enables the anti-nucleolin antibodyto be readily purified by affinity purification using an anti-tagantibody or another type of affinity matrix that binds to the epitopetag. Various tag polypeptides and their respective antibodies are wellknown in the art. A non-limiting summary of example epitope tags thatmay be fused to an anti-nucleolin antibody of the invention is listed inTable 3. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag and itsantibody 12CA5 (Field et al., 1988); the c-myc tag and the 8F9, 3C7,6E10, G4, B7 and 9E10 antibodies thereto (Evan et al., 1985); and theHerpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborskyet at, 1990). Other tag polypeptides include the Flag-peptide (Hopp etal., 1988); the KT3 epitope peptide (Martin et al., 1992); an α-tubulinepitope peptide (Skinner et al., 1991); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth et al., 1990).

TABLE 3 Sequence of Affinity Tags SEQ ID NO: Tag: Amino Acid Sequence: 5 Poly-Arg RRRRR (SEQ ID NO: 5)  6 Poly-Lys KKKK (SEQ ID NO: 6)  7Poly-His HHHHHH (SEQ ID NO: 7)  8 FLAG DYKDDDDK (SEQ ID NO: 8)  9Strep-tag II WSHPQFEK (SEQ ID NO: 9) 10 c-myc EQKLISEEDL (SEQ ID NO: 10)11 S KETAAAKFERQHMDS (SEQ ID NO: 11) 12 HATKDHLIHNVHKEFHAHAHNK (SEQ ID NO: 12) 13 3x FLAGDYKDNDGDYKDHDIDYKDDDDK (SEQ ID NO: 13) 14 Calmodulin-KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 14) binding peptide 15 SBPMDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 15) 16 Chitin-bindingTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPS domainNVPALWQLQ (SEQ ID NO: 16) 17 Glutathione S- Protein transferase 18Maltose-binding Protein protein

In one embodiment, a human anti-nucleolin antibody is linked to ananoparticle. In one embodiment, an isolated human anti-nucleolinmonoclonal antibody is linked to a nanoparticle. In one embodiment, ahuman anti-nucleolin antibody produced by a human B cell is linked to ananoparticle. Cell surface nucleolin has been reported to serve asreceptor for DNA nanoparticles composed of pegylated polylysine and DNA(Chen et al., 2008). In one embodiment, the antibody-nanoparticleconjugate can penetrate a cell expressing nucleolin on its surface morerapidly and extensively than the uncongugated antibody. In oneembodiment, the cell is a cancer cell, tumor cell, virally infectedcell, lymphocyte, or activated lymphocyte.

D. Deposit of Cell Lines

Cell lines that express antibodies that immunospecifically bind one ormore nucleolin (NCL) polypeptides (e.g., SEQ ID NO:4 or fragmentsthereof) were deposited on Nov. 17, 2010, with the American Type CultureCollection (10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A.)as an original deposit and were given the ATCC Deposit Nos. PTA-11495,PTA-11490, PTA-11496, PTA-11491, PTA-11492, PTA-11497, and PTA-11494.The deposit was made under Budapest Treaty, and all restrictions on theavailability to the public of the deposited material will be irrevocablyremoved upon the granting of the patent, except for the requirementsspecified in 3 7 C.P.R. 1.808(b), and the term of the deposit willcomply with 37 C.P.R. 1.806.

V. DIAGNOSTICS

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of cancer cells in a human. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody is used to determine thepresence of cancer cells in a human. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody produced by a human B cell isused to determine the presence of cancer cells in a human.

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of cancer cells in a human sample. In one embodiment, anisolated human anti-nucleolin monoclonal antibody is used to determinethe presence of cancer cells in a human sample. In one embodiment, anisolated anti-nucleolin monoclonal antibody expressed by a human B cellis provided and used to determine the presence of cancer cells in ahuman sample. The term human sample as used refers to biologicalcollections from a subject that include but are not limited to a cell,tissue, plasma, serum, whole blood, sputum, or saliva. In oneembodiment, the detection results from a human subject sample arecompared to those from a control sample. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody is used to determine thepresence of cancer cells in a subject. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody expressed by a human B cell isused to determine the presence of cancer cells in a subject. In oneembodiment the determination is made by binding ahuman anti-nucleolinantibody to nucleolin on the surface of a human cancer cell. The terms“cancer” and “cancerous” refer to or describe a physiological conditionin mammals that is typically characterized by unregulated cell growth.Examples of cancer include, but are not limited to: Acute LymphoblasticLeukemia; Myeloid Leukemia; Acute Myeloid Leukemia; Chronic MyeloidLeukemia; Adrenocortical Carcinoma Adrenocortical Carcinoma;AIDS-Related Cancers; AIDS-Related Lymphoma; Anal Cancer; Astrocytoma,Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Basal CellCarcinoma; Bile Duct Cancer, Extrahepatic; Bladder Cancer; BladderCancer; Bone Cancer, osteosarcoma/Malignant Fibrous Histiocytoma; BrainStem Glioma; Brain Tumor; Brain Tumor, Brain Stem Glioma; Brain Tumor,Cerebellar Astrocytoma; Brain Tumor, Cerebral Astrocytoma/MalignantGlioma; Brain Tumor, Ependymoma; Brain Tumor, Medulloblastoma; BrainTumor, Supratentorial Primitive Neuroectodermal Tumors; Brain Tumor,Visual Pathway and Hypothalamic Glioma; Breast Cancer, Female; BreastCancer, Male; Bronchial Adenomas/Carcinoids; Burkitt's Lymphoma;Carcinoid Tumor; Central Nervous System Lymphoma; CerebellarAstrocytoma; Cerebral Astrocytoma/Malignant Glioma; Cervical Cancer;Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; ChronicMyeloproliferative Disorders; Colon Cancer; Colorectal Cancer; CutaneousT-Cell Lymphoma; B-Cell Lymphoma Endometrial Cancer; Ependymoma;Esophageal Cancer; Esophageal Cancer; Ewing's Family of Tumors;Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor; ExtrahepaticBile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer,Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial; GermCell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma,Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma,Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head andNeck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary);Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma;Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamicand Visual Pathway Glioma; Intraocular Melanoma; Islet Cell Carcinoma(Endocrine Pancreas); Kaposi's Sarcoma; Kidney (Renal Cell) Cancer;Kidney Cancer; Laryngeal Cancer; Leukemia, Acute Lymphoblastic;Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, AcuteMyeloid; Leukemia, Chronic Lymphocytic; Leukemia; Chronic Myelogenous;Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer,Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, SmallCell; Lymphoma, AIDS-Related; Lymphoma, Burkitt's; Lymphoma, CutaneousT-Cell, see Mycosis Fungoides and Sezary Syndrome; Lymphoma, Hodgkin's;Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's; Lymphoma,Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central NervousSystem; Macroglobulinemia, Waldenstrom's; Malignant Fibrous Histiocytomaof Bone/Ostedsarcoma; Medulloblastoma; Melanoma; Melanoma, Intraocular(Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant;Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary;Multiple Endocrine Neoplasia Syndrome; Multiple Myeloma/Plasma CellNeoplasm' Mycosis Fungoides; Myelodysplastic Syndromes;Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia,Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, ChildhoodAcute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; NasalCavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma;Non-Hodgkin's Lymphoma; Non-Hodgkin's Lymphoma During Pregnancy; OralCancer; Oral Cavity Cancer, Lip and; Oropharyngeal Cancer;Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer;Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian LowMalignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer;Pancreatic Cancer, Islet Cell; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Prostate Cancer; Rectal Cancer;Renal Cell (Kidney) Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma;Salivary Gland Cancer; Salivary Gland Cancer; Sarcoma, Ewing's Family ofTumors; Sarcoma, Kaposi's; Sarcoma, Soft Tissue; Sarcoma, Soft Tissue;Sarcoma, Uterine; Sezary Syndrome; Skin Cancer (non-Melanoma); SkinCancer; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small CellLung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Soft TissueSarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma);Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric)Cancer; Stomach (Gastric) Cancer; Supratentorial PrimitiveNeuroectodennal Tumors; T-Cell Lymphoma, Cutaneous, see MycosisFungoides and Sezary Syndrome; Testicular Cancer; Thymoma; Thymoma andThymic Carcinoma; Thyroid Cancer; Thyroid Cancer; Transitional CellCancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational;Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer;Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; VisualPathway and Hypothalamic Glioma; Vulvar Cancer; Waldenstrom'sMacroglobulinemia; and Wilms' Tumor.

In one embodiment, a human anti-nucleolin antibody is used determine thepresence of a proliferative disorder. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody is used determine the presenceof a proliferative disorder. In one embodiment, a human anti-nucleolinantibody produced from a human B cell is used determine the presence ofa proliferative disorder. The terms “cell proliferative disorder,”“proliferative disorder” and “neoplastic disorder,” as used herein,refer to disorders that are associated with some degree of abnormal cellproliferation.

In one embodiment, a human anti-nucleolin antibody is used determine thepresence of a malignant disease wherein nucleolin is expressed on thecell surface or in the cytoplasm. In one embodiment, an isolated humananti-nucleolin monoclonal antibody is used determine the presence of amalignant disease wherein nucleolin is expressed on the cell surface orin the cytoplasm. In one embodiment, an isolated anti-nucleolinmonoclonal antibody expressed by a human B cell is provided and used todetermine the presence of a malignant disease wherein nucleolin isexpressed on the cell surface or in the cytoplasm. The term “malignantdiseases” as used hereing refers to progressive or metastatic diseasesor diseases characterized by small tumor burden such as minimal residualdisease. Examples of malignant diseases that a human anti-nucleolinantibody is used determine the presence of include, but are not limitedto leukemias (e.g., acute myeloid, acute lymphocytic and chronicmyeloid) and cancers (e.g., breast, lung, thyroid or gastrointestinalcancer or a melanoma).

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of a non-malignant cell proliferative disorders whereinnucleolin is expressed on the cell surface or in the cytoplasm. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto determine the presence of a non-malignant cell proliferativedisorders wherein nucleolin is expressed on the cell surface or in thecytoplasm. In one embodiment, an isolated anti-nucleolin monoclonalantibody expressed by a human B cell is provided and used to determinethe presence of a non-malignant cell proliferative disorders whereinnucleolin is expressed on the cell surface or in the cytoplasm. Forexample, specific non-limiting examples of non-malignant cellproliferative disorders that can be used to determine the presence of anon-malignant cell proliferative disorder with an isolated humananti-nucleolin monoclonal antibody include but are not limited to warts,benign prostatic hyperplasia, skin tags, and non-malignant tumors. Forexample, an isolated human anti-nucleolin monoclonal antibody can beused to determine such cell proliferative disorders as benign prostatichyperplasia or unwanted genital warts by targeting the undesirable cellsthat characterize such conditions for removal. In one embodiment, anisolated human anti-nucleolin monoclonal antibody is used determine thepresence of a angiogenic tumor cell wherein nucleolin is expressed onthe cell surface or in the cytoplasm.

In one embodiment, a human anti-nucleolin is to determine the presenceof a tumor. “Tumor”, as used herein, refers to all neoplastic cellgrowth and proliferation, whether malignant or benign, and allpre-cancerous and cancerous cells and tissues.

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of a cell expressing human nucleolin on its surface or inits cytoplasm in a subject with an autoimmune disorder. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto determine the presence of a cell expressing human nucleolin on itssurface or in its cytoplasm in a subject with an autoimmune disorder. Inone embodiment, an isolated anti-nucleolin monoclonal antibody expressedby a human B cell is provided and used to determine the presence of acell expressing human nucleolin on its surface or in its cytoplasm in asubject with an autoimmune disorder. In one embodiment, a humananti-nucleolin antibody is used to determine the presence of a cellexpressing human nucleolin on its surface or in its cytoplasm inlymphocytes. In one embodiment, a isolated human anti-nucleolinmonoclonal antibody is used to determine the presence of a cellexpressing human nucleolin on its surface or in its cytoplasm inlymphocytes. In one embodiment, an isolated human anti-nucleolinmonoclonal antibody expressed by a human B cell is provided and used todetermine the presence of a cell expressing human nucleolin on itssurface or in its cytoplasm in lymphocytes. In one embodiment, thelymphocyte comprises a B cell, T cell, or natural killer cell. In oneembodiment, the lymphocyte comprises a CD4-positive or CD8-positivecells.

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of a cell expressing human nucleolin on its surface or inits cytoplasm in activated lymphocytes or memory cells. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto determine the presence of a cell expressing human nucleolin on itssurface or in its cytoplasm in activated lymphocytes or memory cells. Inone embodiment, an isolated anti-nucleolin monoclonal antibody expressedby a human B cell is provided and used to determine the presence of acell expressing human nucleolin on its surface or in its cytoplasm inactivated lymphocytes or memory cells. In one embodiment, the activatedlymphocyte comprises an activated B cell, T cell, or natural killercell.

The term “autoimmune disease or disorder” refers to a condition in asubject characterized by cellular, tissue and/or organ injury caused byan immunologic reaction of the subject to its own cells, tissues and/ororgans. The term “inflammatory disease” is used interchangeably with theterm “inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders. Exemplary autoimmune diseases ordisorders which may be diagnosed with the use of a human anti-nucleolinantibody include, but are not limited to: alopecia greata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,asthma, autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune oophoritis and orchitis,autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, diabetes, type I diabetes mellitus,diabetic retinopathy, eosinophilic fascites, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, Henoch-Schonlein purpura, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), IgA neuropathy,juvenile arthritis, lichen planus, lupus erthematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1 orimmune-mediated diabetes mellitus, myasthenia gravis, pemphigus-relateddisorders (e.g., pemphigus vulgaris), myelodysplastic syndrome,pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome,Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet'ssyndrome, Still's disease, lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria,Vogt-Koyanagi-Hareda syndrome, chronic inflammatory pneumonitis, andchronic inflammation resulting from chronic viral or bacteriainfections.

In one embodiment, a human anti-nucleolin antibody is used to determinethe presence of a cell expressing human nucleolin on its surface or inits cytoplasm in viral infected cells. In one embodiment, an isolatedhuman anti-nucleolin monoclonal antibody is used to determine thepresence of a cell expressing human nucleolin on its surface or in itscytoplasm in viral infected cells. In one embodiment, an isolatedanti-nucleolin monoclonal antibody expressed by a human B cell isprovided and used determine the presence of a cell expressing humannucleolin on its surface or in its cytoplasm in viral infected cells.Nucleolin is expressed at the cell surface of virus infected cells(Hovanessian et al., 2006; Bose et al., 2004; Izumi et al., 2001).Examples of virus which can infect cells include but are not limited to:Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and otherisolates, such as HIV-LP); Picornaviridae (e.g., polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g., strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Bimaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avianmyeloblastosis virus (AMV)) and C-type group B (including felineleukemia virus (FeLV), gibbon ape leukemia virus (GALV), spleen necrosisvirus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus(SSV)), D-type retroviruses include Mason-Pfizer monkey virus (MPMV) andsimian retrovirus type 1 (SRV-1), the complex retroviruses including thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses, lentiviruses including HIV-1, HIV-2, SIV, Visna virus, felineimmunodeficiency virus (FIV), and equine infectious anemia virus (EIAV),simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV),the foamy viruses including human foamy virus (HFV), simian foamy virus(SFV) and bovine foamy virus (BFV), Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swinefever virus); and unclassified viruses (e.g., the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses), Mycobacterium (Mycobacterium tuberculosis, M. bovis, M.avium-intracellulare, M. leprae), Pneumococcus, Streptococcus,Staphylcococcus, Diphtheria, Listeria, Erysipelothrix, Anthrax, Tetanus,Clostridium, Mixed Anaerobes, Neisseria, Salmonella, Shigella,Hemophilus, Escherichia coli, Klebsiella, Enterobacter, Serratia,Pseudomonas, Bordatella, Francisella tularensis, Yersinia, Vibriocholerae, Bartonella, Legionella, Spirochaetes (Treponema, Leptospira,Borrelia), Fungi, Actinomyces, Rickettsia, Mycoplasma, Chlamydia,Protozoa (including Entamoeba, Plasmodium, Leishmania, Trypanosoma,Toxoplasma, Pneumocystis, Babasia, Giardia, Cryptosporidium,Trichomonas), Helminths (Trichinella, Wucheraria, Onchocerca,Schistosoma, Nematodes, Cestodes, Trematodes), and viral pneumonias.Additional examples of antigens which can be targets for compositions ofthe invention are known, such as those disclosed in U.S. PatentPublication No. 2007/0066554. In another embodiment, a conjugate cancomprise an antigen or cellular component as described herein, but inaddition to a targeting moiety and an immunostimulatory nucleic acidmolecule.

In one embodiment, detectable labels are used in a therapeutic and/ordiagnostic application using an anti-nucleolin antibody. “Detectablelabels” are compounds and/or elements that permit detection of boundantibody. Many appropriate imaging agents are known in the art, as aremethods for their attachment to antibodies (see, for e.g., U.S. Pat.Nos. 5,021,236; 4,938,948; and 4,472,509, each incorporated herein byreference). The imaging moieties used can be paramagnetic ions;radioactive isotopes; fluorochromes; NMR-detectable substances; X-rayimaging agents. Other agents include enzymes, haptens, fluorescentlabels, phosphorescent molecules, chemilluminescent molecules,chromophores, photoaffinity molecules, colored particles or ligands,such as biotin.

In one aspect, the detectable labels comprise paramagnetic ions.Examples of paramagnetic ions include, but are not limited to, chromium(III), manganese (II), iron iron (II), cobalt (II), nickel (II), copper(II), neodymium (III), samarium (III), ytterbium (III), gadolinium(III), vanadium (II), terbium (III), dysprosium (III), holmium (III)and/or erbium (III), with gadolinium being particularly preferred. Ionsuseful in other contexts, such as X-ray imaging, include but are notlimited to lanthanum (III), gold (III), lead (II), and bismuth (III). Inone aspect, the detectable lables comprise radioactive isotopes.Examples of radioactive isotope include, but are not limited to,¹²⁴antimony, ¹²⁵antimony, ⁷⁴arsenic, ²¹¹astatine, ¹⁰³barium, ¹⁴⁰barium,⁷beryllium, ²⁰⁶bismuth, ²⁰⁷bismuth, ¹⁰⁹cadmium, ¹¹⁵cadmium, ⁴⁵calcium,¹⁴carbon, ¹³⁹cerium, ¹⁴¹cerium, ¹⁴⁴cerium, ¹³⁷cesium, ⁵¹chromium,³⁶chlorine, ⁵⁶cobalt, ⁵⁷cobalt, ⁵⁸cobalt, ⁶⁰cobalt, ⁶⁷copper, ¹⁶⁹erbium,¹⁵²eurpium, ⁶⁷gallium, ¹⁵³gadolinium, ¹⁹⁵gold, ¹⁹⁹gold, ¹⁷⁵hafnium,¹⁷⁵⁺¹⁸¹hafnium, ¹⁸¹hafnium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹³¹iodine,¹¹¹indium, ¹⁹²iridium, ⁵⁵iron, ⁵⁹iron, ⁸⁵krypton, ²¹⁰lead, ¹⁷⁷lutecium,⁵⁴manganese, ¹⁹⁷mercury, ²⁰³mercury, ⁹⁹ molybdenum, ¹⁴⁷neodynium,²³⁷neptunium, ⁶³nickel, ⁹⁵niobium, ¹⁸⁵⁺¹⁹¹osmium, ¹⁰³palladium,³²phosphorus, ¹⁸⁴platinum, ¹⁴³praseodymium, ¹⁴⁷promethium, ²³³protactinium ²²⁶radium rhenium¹⁸⁶, ¹⁸⁸rhenium, ⁸⁶rubidium, ¹³⁰ruthenium,¹⁰⁶ruthenium, ⁴⁴scandium, ⁴⁶scandium, ⁴⁵selenium, ⁷⁵selenium,^(110m)silver, ¹¹¹silver, ²²sodium, ⁸⁵strontium, ⁸⁹strontium,⁹⁰strontium, ³⁵sulphur, ¹⁸²tantalum, ^(99m)technicium, ^(125m)tellurium,¹³²tellurium, ¹⁶⁰terbium, ²⁰⁴thallium, ²²⁸thorium, ²³²thorium,¹⁷⁰thullium, ¹¹³tin, ⁴⁴titanium, ¹⁸⁵tungsten, ⁴⁸vanadlum, ⁴⁹vanadium,⁸⁸yttrium, ⁹⁰yttrium, ⁹¹yttrium, ¹⁶⁹ytterbium, ⁶⁵zinc, and ⁹⁵zirconium.

In one embodiment ¹²⁵Iodine technicium^(99m) and/or indium¹¹¹ are useddue to their low energy and suitability for long range detection.Radioactively labeled monoclonal antibodies of the present invention canbe produced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium and/orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Monoclonal antibodies according to the invention can be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column.Alternatively, direct labeling techniques can be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the antibody.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

In one embodiment, a detectable label comprises a fluorescent label.Examples of fluorescent labels include but are not limited to Alexa 350,Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, FluoresceinIsothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, OregonGreen 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red,Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include but are notlimited to urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Particular secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this can not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups can also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter and Haley, 1983).In particular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; Dholakia et al., 1989) and can be used as antibodybinding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can alsobe reacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors isachieved using monoclonal antibodies and the detectable imaging moietiesare bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In one embodiment, the present invention provides a method oftransmitting data from a diagnostic result using a human anti-nucleolinantibody. The diagnostic assay may be used to detect cancer or anautoimmune disorder. An example of transmitting data can be disclosingthe result of any of the methods and assays described herein across theinternet. In one embodiment, the results from diagnostic methods using ahuman anti-nucleolin antibody with biological samples or a subject iscollected and the information is transmitted by digital means, such asby facsimile, electronic mail, telephone, or a global communicationsnetwork, such as the World Wide Web. For example, data can betransmitted via website posting, such as by subscription orselect/secure access thereto and/or via electronic mail and/or viatelephone, IR, radio, television or other frequency signal, and/or viaelectronic signals over cable and/or satellite transmission and/or viatransmission of disks, compact discs (CDs), computers, hard drives, orother apparatus containing the information in electronic form, and/ortransmission of written forms of the information, e.g., via facsimiletransmission and the like. Thus, the invention comprehends a userperforming according to the invention and transmitting informationtherefrom; for instance, to one or more parties who then further utilizesome or all of the data or information, e.g., in the manufacture ofproducts, such as therapeutics, assays and diagnostic tests and etc.This invention comprehends disks, CDs, computers, or other apparatus ormeans for storing or receiving or transmitting data or informationcontaining information from methods and/or use of methods of theinvention. Thus, the invention comprehends a method for transmittinginformation comprising performing a method as discussed herein andtransmitting a result thereof.

In one aspect, the invention provides methods of doing businesscomprising performing or using some or all of the herein methods, andcommunicating or transmitting or divulging a result of a diagnosticassay using a human anti-nucleolin antibody, advantageously in exchangefor compensation, e.g., a fee. Advantageously, the communicating,transmitting or divulging of information is via electronic means, e.g.,via internet or email, or by any other transmission means hereindiscussed. Thus, the invention comprehends methods of doing business.

For example, a first party, a “client,” can request information, e.g.,via any of the herein mentioned transmission means—either previouslyprepared information or information specially ordered as to the resultsof the methods and assays of the invention—of a second party, “vendor”,e.g., requesting information via electronic means such as via internet(for instance request typed into website) or via email. The vendor cantransmit that information, e.g., via any of the transmission meansherein mentioned, advantageously via electronic means, such as internet(for instance secure or subscription or select access website) or email.The information can come from performing some or all of a herein methodor use of a herein method in response to the request, or from performingsome or all of a herein method, and generating a library of informationfrom performing some or all of a herein method or use of a hereinalgorithm. Meeting the request can then be by allowing the client accessto the library or selecting data from the library that is responsive tothe request.

Accordingly, the invention even further comprehends collections ofinformation, e.g., in electronic form (such as forms of transmissiondiscussed above), from performing or using a herein invention.

For example, a client hospital can find itself in need of determiningthe presence of a disease, such as cancer. A vendor proficient at themethods of the present invention can be contacted by the hospital toquickly test or screen a sample form a subject using any of the hereindescribed methods or any other method contemplated by the invention. Theresults of the screen can be transmitted back to the client hospital fora fee.

VI. THERAPEUTIC USE OF MABS

A. Human Monoclonal Anti-Nucleolin Antibodies

In one embodiment, a human anti-nucleolin antibody is provided that canbe used to inhibit or kill a cancer cell. In another embodiment, anisolated human anti-nucleolin monoclonal antibody is provided that canbe used to inhibit or kill a cancer cell. In another embodiment, anisolated anti-nucleolin monoclonal antibody expressed by a human B cellis provided that can be used to inhibit or kill a cancer cell. In oneembodiment the cancer cell expresses human nucleolin on its surface orin it cytoplasm. Examples of cancer cells that can be inhibited orkilled by a human anti-nucleolin antibody include but are not limitedto: Acute Lymphoblastic Leukemia; Myeloid Leukemia; Acute MyeloidLeukemia; Chronic Myeloid Leukemia; Adrenocortical CarcinomaAdrenocortical Carcinoma; AIDS-Related Cancers; AIDS-Related Lymphoma;Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, ChildhoodCerebral; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; BladderCancer; Bladder Cancer; Bone Cancer, osteosarcoma/Malignant FibrousHistiocytoma; Brain Stem Glioma; Brain Tumor; Brain Tumor, Brain StemGlioma; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, CerebralAstrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,Medulloblastoma; Brain Tumor, Supratentorial Primitive NeuroectodermalTumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; BreastCancer, Female; Breast Cancer, Male; Bronchial Adenomas/Carcinoids;Burkitt's Lymphoma; Carcinoid Tumor; Central Nervous System Lymphoma;Cerebellar Astrocytoma; Cerebral Astrocytoma/Malignant Glioma; CervicalCancer; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia;Chronic Myeloproliferative Disorders; Myelodysplastic Syndromes; ColonCancer; Colorectal Cancer; Cutaneous T-Cell Lymphoma; B-Cell LymphomaEndometrial Cancer; Ependymoma; Esophageal Cancer; Esophageal Cancer;Ewing's Family of Tumors; Extracranial Germ Cell Tumor; ExtragonadalGerm Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, IntraocularMelanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric(Stomach) Cancer; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor,Extracranial; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor;Glioma; Glioma, Childhood Brain Stem; Glioma, Childhood CerebralAstrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; HairyCell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer,Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary);Hodgkin's Lymphoma; Hodgkin's Lymphoma During Pregnancy; HypopharyngealCancer; Hypothalamic and Visual Pathway Glioma; Intraocular Melanoma;Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney(Renal Cell) Cancer; Kidney Cancer; Laryngeal Cancer; Leukemia, AcuteLymphoblastic; Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid;Leukemia, Acute Myeloid; Leukemia, Chronic Lymphocytic; Leukemia;Chronic Myelogenous; Lip and Oral Cavity Cancer; Liver Cancer, Adult(Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-SmallCell; Lung Cancer, Small Cell; Lymphoma, AIDS-Related; Lymphoma,Burkitt's; Lymphoma, Cutaneous T-Cell, see Mycosis Fungoides and SezarySyndrome; Lymphoma, Hodgkin's; Lymphoma, Hodgkin's During Pregnancy;Lymphoma, Non-Hodgkin's; Lymphoma, Non-Hodgkin's During Pregnancy;Lymphoma, Primary Central Nervous System; Macroglobulinemia,Waldenstrom's; Malignant Fibrous Histiocytoma of Bone/Osteosarcoma;Medulloblastoma; Melanoma; Melanoma, Intraocular (Eye); Merkel CellCarcinoma; Mesothelioma, Adult Malignant; Mesothelioma; MetastaticSquamous Neck Cancer with Occult Primary; Multiple Endocrine NeoplasiaSyndrome; Multiple Myeloma/Plasma Cell Neoplasm' Mycosis Fungoides;Myelodysplastic Syndromes; Myelodysplastic/Myeloproliferative Diseases;Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; MyeloidLeukemia, Childhood Acute; Myeloma, Multiple; MyeloproliferativeDisorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer;Nasopharyngeal Cancer; Neuroblastoma; Non-Hodgkin's Lymphoma;Non-Hodgkin's Lymphoma During Pregnancy; Oral Cancer; Oral CavityCancer, Lip and; Oropharyngeal Cancer; Osteosarcoma/Malignant FibrousHistiocytoma of Bone; Ovarian Cancer; Ovarian Epithelial Cancer; OvarianGerm Cell Tumor; Ovarian Low Malignant Potential Tumor; PancreaticCancer; Pancreatic Cancer; Pancreatic Cancer, Islet Cell; ParathyroidCancer; Penile Cancer; Pheochromocytoma; Pineoblastoma andSupratentorial Primitive Neuroectodermal Tumors; Pituitary Tumor; PlasmaCell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy andBreast Cancer; Pregnancy and Hodgkin's Lymphoma; Pregnancy andNon-Hodgkin's Lymphoma; Primary Central Nervous System Lymphoma;Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell(Kidney) Cancer; Renal Pelvis and Ureter, Transitional Cell Cancer;Retinoblastoma; Rhabdomyosarcoma; Salivary Gland Cancer; Salivary GlandCancer; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi's; Sarcoma,Soft Tissue; Sarcoma, Soft Tissue; Sarcoma, Uterine; Sezary Syndrome;Skin Cancer (non-Melanoma); Skin Cancer; Skin Cancer (Melanoma); SkinCarcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer;Soft Tissue Sarcoma; Soft Tissue Sarcoma; Squamous Cell Carcinoma, seeSkin Cancer (non-Melanoma); Squamous Neck Cancer with Occult Primary,Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer;Supratentorial Primitive Neuroectodermal Tumors; T-Cell Lymphoma,Cutaneous, see Mycosis Fungoides and Sezary Syndrome; Testicular Cancer;Thymoma; Thymoma and Thymic Carcinoma; Thyroid Cancer; Thyroid Cancer;Transitional Cell Cancer of the Renal Pelvis and Ureter; TrophoblasticTumor, Gestational; Ureter and Renal Pelvis, Transitional Cell Cancer;Urethral Cancer; Uterine Cancer, Endometrial; Uterine Sarcoma; VaginalCancer; Visual Pathway and Hypothalamic Glioma; Vulvar Cancer;Waldenstrom's Macroglobulinemia; and Wilms' Tumor.

In one embodiment, a human anti-nucleolin antibody is used to reducecell viability of a cancer cell in a subject sample by 30 to 80% ascompared to cells not exposed to a human anti-nucleolin antibody. In oneembodiment, an isolated human monoclonal anti-nucleolin antibody is usedto reduce cell viability of a cancer cell in a subject sample by 30 to80% as compared cells not exposed to a human anti-nucleolin antibody. Inone embodiment, an isolated human monoclonal anti-nucleolin antibodyproduced from a human B cell is provided and used to reduce cellviability of a cancer cell in a subject sample by 30 to 80% as comparedcells not exposed to a human anti-nucleolin antibody.

In one embodiment, a human anti-nucleolin antibody is used to reducecell viability of a cancer cell in a subject by 30 to 80% as comparedcells not exposed to a human anti-nucleolin antibody. In one embodiment,an isolated human anti-nucleolin monoclonal antibody is used to reducecell viability of a cancer cell in a subject by 30 to 80% as comparedcells not exposed to a human anti-nucleolin antibody. In one embodiment,an isolated human anti-nucleolin monoclonal antibody produced from ahuman B cell is provided and used to reduce cell viability of a cancercell in a subject by 30 to 80% as compared cells not exposed to a humananti-nucleolin antibody.

In one embodiment a human anti-nucleolin antibody is administered to ahuman subject with one or more forms of cancer. In one embodiment anisolated human anti-nucleolin monoclonal antibody is administered to ahuman subject with one or more forms of cancer. In one embodiment atleast one of the forms of cancer is inhibited or killed by a humananti-nucleolin antibody. In one embodiment an isolated humananti-nucleolin monoclonal antibody is administered to a human subjectwhere the cancer is resistant to other cancer treatments. In oneembodiment an isolated human anti-nucleolin monoclonal antibody producedfrom a human B cell is provided is administered to a human subject wherethe cancer is resistant to other cancer treatments. For example, cancerscan be resistant to radiation therapy, chemotherapy, or biologicaltherapy. In one embodiment the immune system of the human subject ismore tolerant to the isolated human anti-nucleolin antibody than to anisolated non human anti-nucleolin antibody. In another embodiment theimmune system of the human subject is more tolerant to the isolatedhuman anti-nucleolin antibody than to an isolated humanizedanti-nucleolin antibody. In another embodiment the immune system of thehuman subject is more tolerant to the isolated human anti-nucleolinantibody than to an isolated chimeric anti-nucleolin antibody.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill a cell as part of an adjuvant therapy. In one embodiment, anisolated human anti-nucleolin monoclonal antibody is used to inhibit orkill a cell as part of an adjuvant therapy. In one embodiment, anisolated anti-nucleolin monoclonal antibody expressed by a human B cellis provided and used as part of an adjuvant therapy. Adjuvant therapycan include chemotherapy, radiation therapy, hormone therapy, targetedtherapy, or biological therapy. Adjuvant therapy as used herein refersto treatment given after the primary treatment to lower the risk thatthe cancer will come back.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill a cell used in combination with an adjuvant therapy. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto inhibit or kill a cell used in combination with an adjuvant therapy.In one embodiment, an isolated anti-nucleolin monoclonal antibodyexpressed by a human B cell is provided as part of an adjuvant therapy.Adjuvant therapy may include chemotherapy, radiation therapy, hormonetherapy, targeted therapy, or biological therapy.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill a cell of a non-malignant cell proliferative disorder whereinnucleolin is expressed on the cell surface or in the cytoplasm. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto inhibit or kill a cell of a non-malignant cell proliferative disorderwherein nucleolin is expressed on the cell surface or in the cytoplasm.In one embodiment, the isolated anti-nucleolin monoclonal antibody isexpressed by a human B cell. For example, specific non-limiting examplesof non-malignant cell proliferative disorders that can treated orinhibited with an anti-nucleolin antibody include but are not limited towarts, benign prostatic hyperplasia, skin tags, and non-malignanttumors. For example, an isolated human anti-nucleolin monoclonalantibody can be used to determine such cell proliferative disorders asbenign prostatic hyperplasia or unwanted genital warts by targeting theundesirable cells that characterize such conditions for removal.Expression of nucleolin on the cell surface of endothelial cells intumors has been shown to be a unique marker of tumor angiogenesis(Christian et al., 2003). In one embodiment, a human anti-nucleolinantibody is used to inhibit or kill in a subject a cell comprisinganangiogenic tumor. In one embodiment, an isolated human anti-nucleolinmonoclonal antibody is used to inhibit or kill in a subject a cellcomprising an angiogenic tumor. In one embodiment, an isolatedanti-nucleolin monoclonal antibody expressed by a human B cell isprovided that can inhibit or kill in a subject a cell comprising anangiogenic tumor. An angiogenic tumor as used herein a tumor cell with aproliferation of a network of blood vessels that penetrate intocancerous growths, supplying nutrients and oxygen and removing wasteproducts.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill in a subject a tumor cell under conditions of tumor hypoxia. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto inhibit or kill in a subject a tumor cell under conditions of tumorhypoxia. In one embodiment, an isolated anti-nucleolin monoclonalantibody expressed by a human B cell is provided that can inhibit orkill in a subject a tumor cell under conditions of tumor hypoxia. Tumorhypoxia occurs in the situation where tumor cells have been deprived ofoxygen. Tumor hypoxia can be a result of the high degree of cellproliferation undergone in tumor tissue, causing a higher cell density,and thus taxing the local oxygen supply.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill in subject a lymphocyte cell expressing human nucleolin on itssurface. In one embodiment, a isolated human anti-nucleolin monoclonalantibody is used to inhibit or kill in subject a lymphocyte cellexpressing human nucleolin on its surface. In one embodiment, anisolated human anti-nucleolin monoclonal antibody expressed by a human Bcell is provided that is used to inhibit or kill in subject a lymphocytecell expressing human nucleolin on its surface. In one embodiment, thelymphocyte cell comprises a B cell, T cell, or natural killer cell. Inone embodiment, the lymphocyte cell comprises a CD4-positive orCD8-positive cells.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill in a subject an activated lymphocyte or memory cell expressinghuman nucleolin on its surface. In one embodiment, an isolated humananti-nucleolin monoclonal antibody is used to inhibit or kill in asubject an activated lymphocyte or memory cell expressing humannucleolin on its surface. In one embodiment, an isolated anti-nucleolinmonoclonal antibody expressed by a human B cell is provided that is usedto inhibit or kill in subject an activated lymphocyte cell or memorycell expressing human nucleolin on its surface. In a further embodiment,the activated lymphocyte comprises an activated B cell, T cell, ornatural killer cell. In one embodiment, a human anti-nucleolin antibodyis used to inhibit or kill a cell in a subject having an autoimmunedisorder. In one embodiment, an isolated human anti-nucleolin monoclonalantibody is used to inhibit or kill a cell in a subject having anautoimmune disorder. In one embodiment, an isolated anti-nucleolinmonoclonal antibody expressed by a human B cell is provided that is usedto inhibit or kill a cell in a subject having an autoimmune disorder.

In one embodiment, ahuman anti-nucleolin antibody is used to inhibit orkill a cell in a subject having an autoimmune disorder. In oneembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto inhibit or kill a cell in a subject having an autoimmune disorder. Inone embodiment, an isolated anti-nucleolin monoclonal antibody expressedby a human B cell is provided that can inhibit or kill cell in a subjecthaving an autoimmune disorder. CD40 and CD40 ligand are interactionsmediate T-dependent B cell response and efficient T cell priming andnucleolin has been shown to interact with CD40 ligand. (Geahlen et al.,1984) In one embodiment, an isolated human anti-nucleolin monoclonalantibody is used to inhibit or kill a cell in a subject having anautoimmune disorder wherein the cell is characterized by activated CD40receptors. In one embodiment the cell expresses human nucleolin on itssurface or in its cytoplasm. In one embodiment the cell is a lymphocyte.In one embodiment the lymphocyte is a B cell or T cell. In oneembodiment the lymphocyte is activated. Exemplary autoimmune diseases ordisorders which may be diagnosed with the use of a human anti-nucleolinantibody include, but are not limited to: alopecia greata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,asthma, autoimmune diseases of the adrenal gland, autoimmune hemolyticanemia, autoimmune hepatitis, autoimmune oophoritis and orchitis,autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid,cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immunedysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, diabetes, type 1 diabetes mellitus,diabetic retinopathy, eosinophilic fascites, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, Henoch-Schonlein purpura, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), IgA neuropathy,juvenile arthritis, lichen planus, lupus erthematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1 orimmune-mediated diabetes mellitus, myasthenia gravis, pemphigus-relateddisorders (e.g., pemphigus vulgaris), pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still'sdisease, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria,Vogt-Koyanagi-Hareda syndrome, chronic inflammatory pneumonitis, andchronic inflammation resulting from chronic viral or bacteriainfections.

In another embodiment, an human anti-nucleolin antibody is used toinhibit or kill a cell in a subject infected by a virus. In anotherembodiment, an isolated human anti-nucleolin monoclonal antibody is usedto inhibit or kill a cell in a subject infected by a virus. In oneembodiment, an isolated anti-nucleolin monoclonal antibody expressed bya human B cell is provided that can inhibit or kill cell in a subjectinfected by a virus. Examples of virus which can infect cells includebut are not limited to: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP);Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronoviridae(e.g., coronaviruses); Rhabdoviradae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Bimaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus); Rous sarcoma virus (RSV), avian leukemia virus (ALV), and avianmyeloblastosis virus (AMV)) and C-type group B (including felineleukemia virus (FeLV), gibbon ape leukemia virus (GALV), spleen necrosisvirus (SNV), reticuloendotheliosis virus (RV) and simian sarcoma virus(SSV)), D-type retroviruses include Mason-Pfizer monkey virus (MPMV) andsimian retrovirus type 1 (SRV-1), the complex retroviruses including thesubgroups of lentiviruses, T-cell leukemia viruses and the foamyviruses, lentiviruses including HIV-1, HIV-2, SIV, Visna virus, felineimmunodeficiency virus (FIV), and equine infectious anemia virus (EIAV),simian T-cell leukemia virus (STLV), and bovine leukemia virus (BLV),the foamy viruses including human foamy virus (HEV), simian foamy virus(SFV) and bovine foamy virus (BFV), Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g., the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e., Hepatitis C); Norwalk and related viruses, andastroviruses), Mycobacterium (Mycobacterium tuberculosis, M bovis, M.avium-intracellulare, M leprae), Pneumococcus, Streptococcus,Staphylcococcus, Diphtheria, Listeria, Erysipelothrix, Anthrax, Tetanus,Clostridium, Mixed Anaerobes, Neisseria, Salmonella, Shigella,Hemophilus, Escherichia coli, Klebsiella, Enterobacter, Serratia,Pseudomonas, Bordatella, Francisella tularensis, Yersinia, Vibriocholerae, Bartonella, Legionella, Spirochaetes (Treponema, Leptospira,Borrelia), Fungi, Actinomyces, Rickettsia, Mycoplasma, Chlamydia,Protozoa (including Entamoeba, Plasmodium, Leishmania, Trypanosoma,Toxoplasma, Pneumocystis, Babasia, Giardia, Cryptosporidium,Trichomonas), Helminths (Trichinella, Wucheraria, Onchocerca,Schistosoma, Nematodes, Cestodes, Trematodes), and viral pneumonias.Additional examples of antigens which can be targets for compositions ofthe invention are known, such as those disclosed in U.S. PatentPublication No. 2007/0066554. In a further aspect of the invention, aconjugate can comprise an antigen or cellular component as describedherein, but in addition to a targeting moiety and an immunostimulatorynucleic acid molecule.

In one embodiment, a human anti-nucleolin antibody is used to inhibit orkill a cell in a sample from a subject as a indicator for the presenceof a disease. In one embodiment, an isolated human anti-nucleolinmonoclonal antibody is used to inhibit or kill a cell in a sample from asubject as a prognostic indicator for a disease. In one embodiment, anisolated anti-nucleolin monoclonal antibody expressed by a human B cellis provided that is used to inhibit or kill a cell in a sample from asubject as a prognostic indicator for a disease. Examples of diseasestested include but are not limited to malignant tumor, non-malignanttumor, cancer, autoimmune disease, inflammatory disease, and infectiousdisease.

B. Antibody Conjugates

In one embodiment, the present invention provides for an isolated humananti-nucleolin antibody linked to at least one therapeutic agent to forman antibody conjugate. In one embodiment efficacy of an isolated humananti-nucleolin antibody, is linked, or covalently bound, or complexed toat least one therapeutic agent, such as a molecule or moiety.Therapeutic agents comprise molecules having a desired activity, e.g.,cytotoxic activity. In one embodiment a therapeutic agent which can beattached to an antibody includes but is not limited to a toxin (such asa peptide immunotoxin that catalytically inhibit the elongation step ofprotein synthesis) an anti-tumor agent, a therapeutic enzyme, aradionuclide, an antiviral agent, a chelating agent as described herein,a cytokine, a growth factor, or a oligo- or polynucleotide. Conjugationmethodologies are similar to those described above for diagnosticagents.

In one embodiment, an isolated human anti-nucleolin antibody isconjugated to an enzymatically active toxin or fragment thereof.Examples of enzymatically active toxins and fragments thereof include,but are not limited to, diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI,PAPII, and PAP-S), pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins or thetricothecenes.

In another embodiment, an isolated human anti-nucleolin antibody isconjugated to an a radionuclide. Examples of suitable radionuclidesinclude, but are not limited to, ¹²⁴antimony, ¹²⁵antimony, ⁷⁴arsenic,²¹¹astatine, ¹⁰³barium, ¹⁴⁰barium, ⁷beryllium, ²⁰⁶bismuth, ²⁰⁷bismuth,¹⁰⁹cadmium, ¹¹⁵cadmium, ⁴⁵calcium, ¹⁴carbon, ¹³⁹cerium, ¹⁴¹cerium,¹⁴⁴cerium, ¹³⁷cesium, ⁵¹chromium, ³⁶chlorine, ⁵⁶cobalt, ⁵⁷cobalt,⁵⁸cobalt, ⁶⁰cobalt, ⁶⁷copper, ¹⁶⁹erbium, ¹⁵²eurpium, ⁶⁷gallium,¹⁵³gadolinium, ¹⁹⁵gold, ¹⁹⁹gold, ¹⁷⁵hafnium, ¹⁷⁵⁺¹⁸¹hafnium, ¹⁸¹hafnium,³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹³¹iodine, ¹¹¹indium, ¹⁹²iridium,⁵⁵iron, ⁵⁹iron, ⁸⁵krypton, ²¹⁰lead, ¹⁷⁷lutecium, ⁵⁴manganese,¹⁹⁷mercury, ²⁰³mercury, ⁹⁹ molybdenum, ¹⁴⁷neodynium, ²³⁷neptunium,⁶³nickel, ⁹⁵niobium, ¹⁸⁵⁺¹⁹¹osmium, ¹⁰³palladium, ³²phosphorus,¹⁸⁴platinum, ¹⁴³praseodymium, ¹⁴⁷promethium, ²³³ protactinium ²²⁶radiumrhenium¹⁸⁶, ¹⁸⁸rhenium, ⁸⁶rubidium, ¹³⁰ruthenium, ¹⁰⁶ruthenium,⁴⁴scandium, ⁴⁶scandium, ⁴⁵selenium, ⁷⁵selenium, ^(110m)silver,¹¹¹silver, ²²sodium, ⁸⁵strontium, ⁸⁹strontium, ⁹⁰strontium, ³⁵sulphur,¹⁸²tantalum, ^(99m)technicium, ^(125m)tellurium, ¹³²tellurium,¹⁶⁰terbium, ²⁰⁴thallium, ²²⁸thorium, ²³²thorium, ¹⁷⁰thullium, ¹¹³tin,⁴⁴titanium, ¹⁸⁵tungsten, ⁴⁸vanadlum, ⁴⁹vanadium, ⁸⁸yttrium, ⁹⁰yttrium,⁹¹yttrium, ¹⁶⁹ytterbium, ⁶⁵zinc, and ⁹⁵zirconium.

Conjugates of the antibody and cytotoxic agent can be made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO 94/11026.

In one embodiment, an isolated human anti-nucleolin antibody isconjugated to an a cytokine. The term “cytokine” is a generic term forproteins released by one cell population which act on another cell asintercellular mediators. Examples of such cytokines are lymphokines,monokines, and traditional polypeptide hormones. Included among thecytokines are growth hormone such as human growth hormone, N-methionylhuman growth hormone, and bovine growth hormone; parathyroid hormone;thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoproteinhormones such as follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), and luteinizing hormone (LH); hepatic growth factor;fibroblast growth factor; prolactin; placental lactogen; tumor necrosisfactor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

In another embodiment, an isolated human anti-nucleolin antibody isconjugated to an chemotherapeutic agent. A variety of chemicalcompounds, also described as “chemotherapeutic agents,” function toinduce DNA damage. Categories of chemotherapeutic agents suitable forconjugation with a an isolated human anti-nucleolin antibody include,but are not limited to, alkylating agents, anthracyclines, cytoskeletaldisruptors, epothilones, inhibitors of topoisomerase I, inhibitors oftopoisomerase II, nucleoside and nucleotide analogs and precursoranalogs, peptide antibiotics, platinum-based agents, retinoids, or vincaalkaloids and derivatives. Specific chemotherapeutic agents within thesegroups include, but are not limited to, actinomycin-D, all-transretinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,vinorelbine. The invention also encompasses the use of a combination ofone or more DNA damaging agents, whether radiation-based or actualcompounds, such as the use of X-rays with cisplatin or the use ofcisplatin with etoposide.

In another embodiment, an isolated human anti-nucleolin antibody isconjugated to an anti-viral agent. Example of anti-viral agents that canbe used with an isolated human anti-nucleolin antibody include, but arenot limited to, substrates and substrate analogs, inhibitors and otheragents that severely impair, debilitate or otherwise destroyvirus-infected cells. Substrate analogs include amino acid andnucleoside analogs. Substrates can be conjugated with toxins or otherviricidal substances. Inhibitors include integrase inhibitors, proteaseinhibitors, polymerase inhibitors and transcriptase inhibitors such asreverse transcriptase inhibitors.

Specific antiviral agents that can be used with an isolated humananti-nucleolin antibody include, but are not limited to, ganciclovir,valganciclovir, oseltamivir (Tamiflu), zanamivir (Relenza), abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz,emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen,fosamprenavir, foscarnet, fosfonet, fusion inhibitors (e.g.,enfuvirtide), ibacitabine, immunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitor, interferon type III, interferon type II,interferon type I, interferon, lamivudine, lopinavir, loviride,maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleosideanalogues, peginterferon alfa-2a, penciclovir, peramivir, pleconaril,podophyllotoxin, protease inhibitor, raltegravir, reverse transcriptaseinhibitor, ribavirin, rimantadine, ritonavir, pyrimidine antiviral,saquinavir, stavudine, synergistic enhancer (antiretroviral), tenofovir,tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine,truvada, valaciclovir (Valtrex), vicriviroc, vidarabine, viramidine,zalcitabine, and zidovudine.

Examples of nucleoside analogs that can be used with an isolated humananti-nucleolin antibody include acyclovir (ACV), ganciclovir (GCV),famciclovir, foscarnet, ribavirin, zalcitabine (ddC), zidovudine (AZT),stavudine (D4T), lamivudine (3TC), didanosine (ddI), cytarabine,dideoxyadenosine, edoxudine, floxuridine, idozuridine, inosine pranobex,2′-deoxy-5-(methylamino)uridine, trifluridine and vidarabine.

C. Pharmaceutical Compositions and Administration

It is envisioned that, for administration to a subject in need thereof,a antibody will be suspended in a formulation suitable foradministration to a host. In one embodiment the antibody is a monoclonalantibody. In one embodiment the monoclonal antibody is an anti-nucleolinantibody. In one embodiment the monoclonal anti-nucleolin antibody is ahuman monoclonal anti-nucleolin antibody. Aqueous compositions of thepresent invention comprise an effective amount of an antibody dispersedin a pharmaceutically acceptable formulation and/or aqueous medium. Thephrases “pharmaceutically and/or pharmacologically acceptable” refer tocompositions that do not produce an adverse, allergic and/or otheruntoward reaction when administered to an animal, and specifically tohumans, as appropriate.

As used herein, “pharmaceutically acceptable carrier” includes anysolvents, dispersion media, coatings, antibacterial and/or antifungalagents, isotonic and/or absorption delaying agents and the like. The useof such media or agents for pharmaceutical active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. For administration to humans,preparations should meet sterility, pyrogenicity, general safety and/orpurity standards as required by FDA Office of Biologics standards.

In one embodiment, a human anti-nucleolin antibody of the invention canbe administered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. Any of these molecules can be administered to a patient alone, orin combination with other agents, drugs or hormones, in pharmaceuticalcompositions where it is mixed with suitable excipient(s), adjuvants,and/or pharmaceutically acceptable carriers. In one embodiment of thepresent invention, the pharmaceutically acceptable carrier ispharmaceutically inert.

Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (e.g., directly to a tumor), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. In addition to the activeingredients, these pharmaceutical compositions can contain suitablepharmaceutically acceptable carriers comprising excipients and othercompounds that facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Further details ontechniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Maack Publishing Co,Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, etc., suitablefor ingestion by the patient. See PCT publication WO 93/23572.

Pharmaceutical preparations for oral use may be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable additional compounds, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers include, but are not limited to sugars, including lactose,sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato,or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage).

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders such aslactose or starches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the active compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner similar to that known in the art (e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses).

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Salts tend to be more soluble in aqueous orother protonic solvents that are the corresponding free base forms. Inother cases, the preparation may be a lyophilized powder in 1 mM-50 mMhistidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5,that is combined with buffer prior to use.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

After pharmaceutical compositions comprising a compound of the inventionformulated in an acceptable carrier have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of human telomerase proteins andnucleic acids, such labeling would include amount, frequency and methodof administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. “Therapeuticallyeffective amount” or “pharmacologically effective amount” are wellrecognized phrases and refer to that amount of an agent effective toproduce the intended pharmacological result. Thus, a therapeuticallyeffective amount is an amount sufficient to ameliorate the symptoms ofthe disease being treated. One useful assay in ascertaining an effectiveamount for a given application (e.g., a therapeutically effectiveamount) is measuring the effect on cell survival. The amount actuallyadministered will be dependent upon the individual to which treatment isto be applied, and will preferably be an optimized amount such that thedesired effect is achieved without significant side-effects.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in any appropriate animalmodel. The animal model is also used to achieve a desirableconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans.

In an animal, a “therapeutically effective amount” is the quantity ofcompound which results in an improved clinical outcome as a result ofthe treatment compared with a typical clinical outcome in the absence ofthe treatment. An “improved clinical outcome” refers, for, example, to alonger life expectancy, fewer complications, fewer symptoms, lessphysical discomfort and/or fewer hospitalizations as a result of thetreatment. Improved clinical outcome can be quantified as a certainpercent of subjects receiving administration and improving in theirdisease state over certain period of time. The certain percent ofsubjects receiving administration and improving in their disease statemay be about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The certainpercent of subjects receiving administration and improving in theirdisease state may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or85%. The certain percent of subjects receiving administration andimproving in their disease state may be about 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The certainperiod of time to measure improved clinical outcome may be 1, 2, 3, 4,5, 6, or 7 days. The certain period of time to measure improved clinicaloutcome may be 1, 2, 3, or 4 weeks. The certain period of time tomeasure improved clinical outcome may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more years.

With respect to cancer, an “improved clinical outcome” includes a longerlife expectancy. It can also include slowing or arresting the rate ofgrowth of a tumor, causing a shrinkage in the size of the tumor, adecreased rate of metastasis or an improved quality of life (e.g., adecrease in physical discomfort or an increase in mobility).

With respect to modulation of the immune system, “an improved clinicaloutcome” refers to an increase in the magnitude of the immune responsein the individual, if the individual has a disease involving immunesuppression. “An improved clinical outcome” for individuals withsuppressed immune systems can also refer to a lesser susceptibility toinfectious diseases. For diseases involving an overactive immune system,“an improved clinical outcome” can refer to a decrease in the magnitudeof the immune response. In both cases, an improved clinical outcome canalso involve an improvement in the quality of life, as described above.

A therapeutically effective amount refers to that amount of protein,polypeptide, peptide, antibody, oligo- or polynucleotide, agonist orantagonists which ameliorates the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals(e.g., ED₅₀, the dose therapeutically effective in 50% of thepopulation; and LD₅₀, the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, ED₅₀/LD₅₀ Pharmaceuticalcompositions which exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies is used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state (e.g., tumor size and location; age,weight and gender of the patient; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy). Administration may be every day, everyother day, every week, every other week, every month, every other month,or any variation thereof. Administration of a dosage form comprising ahuman anti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 days. Administration of a dosage form comprising a humananti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 weeks. Administration of a dosage form comprising a humananti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 months. Administration of a dosage form comprising a humananti-nucleolin antibody may be for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12 years. Administration of one or more agents (e.g., a humananti-nucleolin antibody and an other agent) can be intermittent; forexample, administration can be once every two days, every three days,every five days, once a week, once or twice a month, and the like. Longacting pharmaceutical compositions may be administered every 3 to 4days, every week, or once every two weeks depending on half-life andclearance rate of the particular formulation. Guidance as to particulardosages and methods of delivery is provided in the literature (see, U.S.Pat. Nos. 4,657,760; 5,206,344; and 5,225,212, herein incorporated byreference). In one embodiment, the dosage of a composition comprisinghuman anti-nucleolin antibody is administered to a patient is about 0.1mg/kg to 500 mg/kg of the patient's body weight. The amount, forms,and/or amounts of the different forms can be varied at different timesof administration. In one embodiment, a human anti-nucleolin antibody isadministered to a subject. The subjects can be male or female and may beof any race or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectscan be of any age, including newborn, neonate, infant, child,adolescent, adult, and geriatric. Subjects can also include animalsubjects, particularly mammalian subjects such as dog, cat, horse,mouse, rat, etc., screened for veterinary medicine or pharmaceuticaldrug development purposes. Subjects further include, but are notlimited, to those who have, have been exposed to, or have beenpreviously diagnosed as afflicted with a proliferative disorder, such ascancer, or an autoimmune disorder, such as a viral disorder such as HIVor AIDS.

D. Nucleolin-Expressing Cancers and Non-Malignant Cells

In one embodiment, an isolated human anti-nucleolin antibody produced inaccordance with the present invention is used in treating a variety ofcells, including both cancerous and non-cancerous cells. In oneembodiment the isolated human anti-nucleolin antibody is a monoclonalantibody. In another embodiment the isolated human anti-nucleolinantibody is a polyclonal antibody. The term “cancer” is describedpreviously herein. Examples of types cancer that can be inhibited ortreated with an isolated human anti-nucleolin antibody include, but arenot limited to: Acute Lymphoblastic Leukemia; Myeloid Leukemia; AcuteMyeloid Leukemia; Chronic Myeloid Leukemia; Adrenocortical CarcinomaAdrenocortical Carcinoma; AIDS-Related Cancers; AIDS-Related Lymphoma;Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, ChildhoodCerebral; Basal Cell Carcinoma; Bile Duct Cancer, Extrahepatic; BladderCancer; Bladder Cancer; Bone Cancer, osteosarcoma/Malignant FibrousHistiocytoma; Brain Stem Glioma; Brain Tumor; Brain Tumor, Brain StemGlioma; Brain Tumor, Cerebellar Astrocytoma; Brain Tumor, CerebralAstrocytoma/Malignant Glioma; Brain Tumor, Ependymoma; Brain Tumor,Medulloblastoma; Brain Tumor, Supratentorial Primitive NeuroectodermalTumors; Brain Tumor, Visual Pathway and Hypothalamic Glioma; BreastCancer, Female; Breast Cancer, Male; Bronchial Adenomas/Carcinoids;Burkitt's Lymphoma; Carcinoid Tumor; Central Nervous System Lymphoma;Cerebellar Astrocytoma; Cerebral Astrocytoma/Malignant Glioma; CervicalCancer; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia;Chronic Myeloproliferative Disorders; Colon Cancer; Colorectal Cancer;Cutaneous T-Cell Lymphoma; B-Cell Lymphoma Endometrial Cancer;Ependymoma; Esophageal Cancer; Esophageal Cancer; Ewing's Family ofTumors; Extracranial Germ Cell Tumor; Extragonadal Germ Cell Tumor;Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; EyeCancer, Retinoblastoma; Gallbladder Cancer, Gastric (Stomach) Cancer;Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial; GermCell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma; Glioma,Childhood Brain Stem; Glioma, Childhood Cerebral Astrocytoma; Glioma,Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head andNeck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary);Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma;Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamicand Visual Pathway Glioma; Intraocular Melanoma; Islet Cell Carcinoma(Endocrine Pancreas); Kaposi's Sarcoma; Kidney (Renal Cell) Cancer;Kidney Cancer; Laryngeal Cancer; Leukemia, Acute Lymphoblastic;Leukemia, Acute Lymphoblastic; Leukemia, Acute Myeloid; Leukemia, AcuteMyeloid; Leukemia, Chronic Lymphocytic; Leukemia; Chronic Myelogenous;Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer,Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, SmallCell; Lymphoma, AIDS-Related; Lymphoma, Burkitt's; Lymphoma, CutaneousT-Cell, see Mycosis Fungoides and Sezary Syndrome; Lymphoma, Hodgkin's;Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin's; Lymphoma,Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central NervousSystem; Macroglobulinemia, Waldenstrom's; Malignant Fibrous Histiocytomaof Bone/Osteosarcoma; Medulloblastoma; Melanoma; Melanoma, Intraocular(Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant;Mesothelioma; Metastatic Squamous Neck Cancer with Occult Primary;Multiple Endocrine Neoplasia Syndrome; Multiple Myeloma/Plasma CellNeoplasm' Mycosis Fungoides; Myelodysplastic Syndromes;Myelodysplastic/Myeloproliferative Diseases; Myelogenous Leukemia,Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, ChildhoodAcute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; NasalCavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Neuroblastoma;Non-Hodgkin's Lymphoma; Non-Hodgkin's Lymphoma During Pregnancy; OralCancer; Oral Cavity Cancer, Lip and; Oropharyngeal Cancer;Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer;Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian LowMalignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer;Pancreatic Cancer, Islet Cell; Parathyroid Cancer; Penile Cancer;Pheochromocytoma; Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors; Pituitary Tumor; Plasma Cell Neoplasm/MultipleMyeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer;Pregnancy and Hodgkin's Lymphoma; Pregnancy and Non-Hodgkin's Lymphoma;Primary Central Nervous System Lymphoma; Prostate Cancer; Rectal Cancer;Renal Cell (Kidney) Cancer; Renal Cell (Kidney) Cancer; Renal Pelvis andUreter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma;Salivary Gland Cancer; Salivary Gland Cancer; Sarcoma, Ewing's Family ofTumors; Sarcoma, Kaposi's; Sarcoma, Soft Tissue; Sarcoma, Soft Tissue;Sarcoma, Uterine; Sezary Syndrome; Skin Cancer (non-Melanoma); SkinCancer; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small CellLung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma; Soft TissueSarcoma; Squamous Cell Carcinoma, see Skin Cancer (non-Melanoma);Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric)Cancer; Stomach (Gastric) Cancer; Supratentorial PrimitiveNeuroectodermal Tumors; T-Cell Lymphoma, Cutaneous, see MycosisFungoides and Sezary Syndrome; Testicular Cancer; Thymoma; Thymoma andThymic Carcinoma; Thyroid Cancer; Thyroid Cancer; Transitional CellCancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational;Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer;Uterine Cancer, Endometrial; Uterine Sarcoma; Vaginal Cancer; VisualPathway and Hypothalamic Glioma; Vulvar Cancer; Waldenstrom'sMacroglobulinemia; and Wilms' Tumor.

Cancer cells known to express nucleolin include lung cancers (e.g.,non-small cell lung cancers), breast cancers, prostate cancers, coloncancers, pancreatic cancers, renal cell carcinomas, ovarian cancers,leukemias (e.g., AML, CLL), melanomas, glioblastomas, neuroblastomas,sarcomas and gastric cancers. In addition, non-cancer cells that expressnucleolin include immune cells such as dendritic cells, peripheral bloodmonocytes, macrophages, and glial cells, as well as vascular smoothmuscle cells and endothelial cells. In one embodiment, a antibody of thepresent invention is used in a treatment for subjects with hyper-immuneand hyper-angiogenic diseases, the latter being described in U.S. PatentPublication No. 2009/0191244, incorporated herein by reference.

i. Acute Myeloid Leukemia

Acute myeloid leukemia (AML), also known as acute myelogenous leukemia,is a cancer of the myeloid line of blood cells, characterized by therapid growth of abnormal white blood cells that accumulate in the bonemarrow and interfere with the production of normal blood cells. AML isthe most common acute leukemia affecting adults, and its incidenceincreases with age. Although AML is a relatively rare disease,accounting for approximately 1.2% of cancer deaths in the United States,its incidence is expected to increase as the population ages.

The symptoms of AML are caused by replacement of normal bone marrow withleukemic cells, which causes a drop in red blood cells, platelets, andnormal white blood cells. These symptoms include fatigue, shortness ofbreath, easy bruising and bleeding, and increased risk of infection.Although several risk factors for AML have been identified, the specificcause of the disease remains unclear. As an acute leukemia, AMLprogresses rapidly and is typically fatal within weeks or months if leftuntreated.

AML has several subtypes; treatment and prognosis varies among subtypes.Five-year survival varies from 5-70%, and relapse rate varies from30-95%, depending on subtype. AML is treated initially with chemotherapyaimed at inducing a remission; patients can go on to receive additionalchemotherapy or a hematopoietic stem cell transplant. Recent researchinto the genetics of AML has developed tests that better predict howlong a patient is likely to survive and whether a drug is likely to beeffective.

The first symptom leading to a diagnosis of AML is typicallynon-specific, related to one or more of the cytopenias, e.g., anemia,neutropenia, and/or thrombocytopenia. While an excess of abnormal whiteblood cells (leukocytosis) with immature blood cells (blasts) in theperipheral blood are a common finding, and leukemic blasts are sometimesseen, AML can also present with isolated decreases in platelets, redblood cells, or even with a low white blood cell count (leukopenia).While a presumptive diagnosis of AML can be made via examination of theperipheral blood smear when there are circulating leukemic blasts, adefinitive diagnosis usually requires an examination of the cells takenfrom a bone marrow aspiration and biopsy.

Marrow or blood is examined via light microscopy as well as flowcytometry to diagnose the presence of leukemia, to differentiate AMLfrom other types of leukemia (e.g., acute lymphoblastic leukemia), andto classify the subtype of disease (see below). A sample of marrow orblood is typically also tested for various chromosomal aberrations byroutine cytogenetics or fluorescent in situ hybridization. Geneticstudies can also be performed to look for specific mutations in genessuch as FLT3, nucleophosmin, and bcr/able among others, which caninfluence the outcome of the disease.

Cytochemical stains on blood and bone marrow smears are helpful in thedistinction of AML from ALL and in subclassification of AML. Thecombination of a myeloperoxidase or Sudan black stain and a non specificesterase stain will provide the desired information in most cases. Themyeloperoxidase or Sudan black reactions are most useful in establishingthe identity of AML and distinguishing from ALL. The non-specificesterase stain is used to identify a monocytic component in AMLs and todistinguish a poorly differentiated monoblastic leukemia from ALL.

The diagnosis and classification of AML can be challenging, and shouldbe performed by a qualified hematopathologist or hematologist. Instraightforward cases, the presence of certain morphologic features(such as Auer rods) or specific flow cytometry results can distinguishAML from other leukemias; however, in the absence of such features,diagnosis can be more difficult.

According to the widely used WHO criteria, the diagnosis of AML isestablished by demonstrating involvement of more than 20% of the bloodand/or bone marrow by leukemic myeloblasts. AML must be carefullydifferentiated from “pre-leukemic” conditions such as myelodysplastic ormyeloproliferative syndromes, which are treated differently.

Because acute promyelocytic leukemia (APL) has the highest curabilityand requires a unique form of treatment, it is important to quicklyestablish or exclude the diagnosis of this subtype of leukemia.Fluorescent in situ hybridization performed on blood or bone marrow isoften used for this purpose, as it readily identifies the chromosomaltranslocation (t[15;17]) that characterizes APL.

The malignant cell in AML is the myeloblast. In normal hematopoiesis,the myeloblast is an immature precursor of myeloid white blood cells; anormal myeloblast will gradually mature into a mature white blood cell.However, in AML, a single myeloblast accumulates genetic changes which“freeze” the cell in its immature state and prevent differentiation.Such a mutation alone does not cause leukemia; however, when such a“differentiation arrest” is combined with other mutations which disruptgenes controlling proliferation, the result is the uncontrolled growthof an immature clone of cells, leading to the clinical entity of AML.

Much of the diversity and heterogeneity of AML stems from the fact thatleukemic transformation can occur at a number of different steps alongthe differentiation pathway. Modern classification schemes for AMLrecognize that the characteristics and behavior of the leukemic cell(and the leukemia) can depend on the stage at which differentiation washalted.

Specific cytogenetic abnormalities can be found in many patients withAML; the types of chromosomal abnormalities often have prognosticsignificance. The chromosomal translocations encode abnormal fusionproteins, usually transcription factors whose altered properties cancause the “differentiation arrest.” For example, in acute promyelocyticleukemia, the t(15;17) translocation produces a PML-RARα fusion proteinwhich binds to the retinoic acid receptor element in the promoters ofseveral myeloid-specific genes and inhibits myeloid differentiation.

The clinical signs and symptoms of AML result from the fact that, as theleukemic clone of cells grows, it tends to displace or interfere withthe development of normal blood cells in the bone marrow. This leads toneutropenia, anemia, and thrombocytopenia. The symptoms of AML are inturn often due to the low numbers of these normal blood elements. Inrare cases, patients can develop a chloroma, or solid tumor of leukemiccells outside the bone marrow, which can cause various symptomsdepending on its location.

Treatment of AML consists primarily of chemotherapy, and is divided intotwo phases: induction and postremission (or consolidation) therapy. Thegoal of induction therapy is to achieve a complete remission by reducingthe amount of leukemic cells to an undetectable level; the goal ofconsolidation therapy is to eliminate any residual undetectable diseaseand achieve a cure.

All FAB subtypes except M3 are usually given induction chemotherapy withcytarabine (ara-C) and an anthracycline (such as daunorubicin oridarubicin). This induction chemotherapy regimen is known as “7+3” (or“3+7”), because the cytarabine is given as a continuous IV infusion forseven consecutive days while the anthracycline is given for threeconsecutive days as an IV push. Up to 70% of patients will achieve aremission with this protocol. Other alternative induction regimens,including high-dose cytarabine alone or investigational agents, can alsobe used. Because of the toxic effects of therapy, includingmyelosuppression and an increased risk of infection, inductionchemotherapy can not be offered to the very elderly, and the options caninclude less intense chemotherapy or palliative care.

The M3 subtype of AML, also known as acute promyelocytic leukemia, isalmost universally treated with the drug ATRA (all-trans-retinoic acid)in addition to induction chemotherapy. Care must be taken to preventdisseminated intravascular coagulation (DIC), complicating the treatmentof APL when the promyelocytes release the contents of their granulesinto the peripheral circulation. APL is eminently curable withwell-documented treatment protocols.

The goal of the induction phase is to reach a complete remission.Complete remission does not mean that the disease has been cured;rather, it signifies that no disease can be detected with availablediagnostic methods. Complete remission is obtained in about 50%-75% ofnewly diagnosed adults, although this can vary based on the prognosticfactors described above. The length of remission depends on theprognostic features of the original leukemia. In general, all remissionswill fail without additional consolidation therapy.

Even after complete remission is achieved, leukemic cells likely remainin numbers too small to be detected with current diagnostic techniques.If no further post-remission or consolidation therapy is given, almostall patients will eventually relapse. Therefore, more therapy isnecessary to eliminate non-detectable disease and prevent relapse—thatis, to achieve a cure.

The specific type of postremission therapy is individualized based on apatient's prognostic factors (see above) and general health. Forgood-prognosis leukemias (i.e., inv(16), t(8;21), and t(15;17)),patients will typically undergo an additional 3-5 courses of intensivechemotherapy, known as consolidation chemotherapy. For patients at highrisk of relapse (e.g. those with high-risk cytogenetics, underlying MDS,or therapy-related AML), allogeneic stem cell transplantation is usuallyrecommended if the patient is able to tolerate a transplant and has asuitable donor. The best postremission therapy for intermediate-risk AML(normal cytogenetics or cytogenetic changes not falling into good-riskor high-risk groups) is less clear and depends on the specificsituation, including the age and overall health of the patient, thepatient's personal values, and whether a suitable stem cell donor isavailable.

Despite aggressive therapy, however, only 20%-30% of patients enjoylong-term disease-free survival. For patients with relapsed AML, theonly proven potentially curative therapy is a stem cell transplant, ifone has not already been performed. In 2000, the monoclonalantibody-linked cytotoxic agent gemtuzumab ozogamicin (Mylotarg) wasapproved in the United States for patients aged more than 60 years withrelapsed AML who are not candidates for high-dose chemotherapy.

Patients with relapsed AML who are not candidates for stem celltransplantion, or who have relapsed after a stem cell transplant, can beoffered treatment in a clinical trial, as conventional treatment optionsare limited. Agents under investigation include cytotoxic drugs such asclofarabine as well as targeted therapies such as farnesyl transferaseinhibitors, decitabine, and inhibitors of MDR1 (multidrug-resistanceprotein). Since treatment options for relapsed AML are so limited,another option which can be offered is palliative care.

For relapsed acute promyelocytic leukemia (APL), arsenic trioxide hasbeen tested in trials and approved by the Food and Drug Administration.Like ATRA, arsenic trioxide does not work with other subtypes of AML.

Acute myeloid leukemia is a curable disease; the chance of cure for aspecific patient depends on a number of prognostic factors. The singlemost important prognostic factor in AML is cytogenetics, or thechromosomal structure of the leukemic cell. Certain cytogeneticabnormalities are associated with very good outcomes (for example, thet(15;17) translocation in acute promyelocytic leukemia). About half ofAML patients have “normal” cytogenetics; they fall into an intermediaterisk group. A number of other cytogenetic abnormalities are known toassociate with a poor prognosis and a high risk of relapse aftertreatment.

AML which arises from a pre-existing myelodysplastic syndrome ormyeloproliferative disease (so-called secondary AML) has a worseprognosis, as does treatment-related AML arising after chemotherapy foranother previous malignancy. Both of these entities are associated witha high rate of unfavorable cytogenetic abnormalities.

In some studies, age >60 years and elevated lactate dehydrogenase levelwere also associated with poorer outcomes. As with most forms of cancer,performance status (i.e., the general physical condition and activitylevel of the patient) plays a major role in prognosis as well.

FLT3 internal tandem duplications (ITDs) have been shown to confer apoorer prognosis in AML. Treating these patients with more aggressivetherapy, such as stem-cell transplantation in first remission, has notbeen shown to enhance long-term survival, so this prognostic feature isof uncertain clinical significance at this point. ITDs of FLT3 can beassociated with leukostasis.

Researchers are investigating the clinical significance of c-KITmutations in AML. These are prevalent, and clinically relevant becauseof the availability of tyrosine kinase inhibitors, such as imatinib andsunitinib that can block the activity of c-KIT pharmacologically. Othergenes being investigated as prognostic factors or therapeutic targetsinclude CEBPA, BAALC, ERG, and NPM1.

Cure rates in clinical trials have ranged from 20-45%; however, itshould be noted that clinical trials often include only younger patientsand those able to tolerate aggressive therapies. The overall cure ratefor all patients with AML (including the elderly and those unable totolerate aggressive therapy) is likely lower. Cure rates forpromyelocytic leukemia can be as high as 98%.

ii. Chronic Lymphocytic Leukemia

B-cell chronic lymphocytic leukemia (B-CLL), also known as chroniclymphoid leukemia (CLL), is the most common type of leukemia. Leukemiasare abnormal and malignant neoplastic proliferations of the white bloodcells (leukocytes). CLL involves a particular subtype of white bloodcells, which is a lymphocyte called a B cell. B cells originate in thebone marrow, develop in the lymph nodes, and normally fight infection.In CLL, the DNA of a B cell is damaged, so that it can't fight infectionby producing antibodies. Additionally, they grow out of control andaccumulate in the bone marrow and blood, where they crowd out healthyblood cells.

CLL is a disease of adults, but in rare cases it can occur in teenagersand occasionally in children (inherited). Most (>75%) people newlydiagnosed with CLL are over the age of 50, and the majority are men.Most people are diagnosed without symptoms as the result of a routineblood test that returns a high white blood cell count, but as itadvances CLL results in swollen lymph nodes, spleen, and liver, andeventually anemia and infections. Early CLL is not treated, and late CLLis treated with chemotherapy and monoclonal antibodies. Survival variesfrom 5 years to more than 25 years. It is now possible to predictsurvival length more precisely by examining the DNA mutations; patientswith slowly-progressing disease can be reassured and can not need anytreatment in their lifetimes.

Although not originally appreciated, CLL is now felt to be identical toa disease called small lymphocytic lymphoma (SLL), a type ofnon-Hodgkin's lymphoma which presents primarily in the lymph nodes. TheWorld Health Organization considers CLL and SLL to be one disease atdifferent stages, not two separate entities.

Staging, determining the extent of the disease, is done with the Raistaging system or the Binet classification and is based primarily on thepresence, or not, of a low platelet or red cell count. Early stagedisease does not need to be treated.

Recent publications suggest that two or three prognostic groups of CLLexist based on the maturational state of the cell. This distinction isbased on the maturity of the lymphocytes as discerned by theimmunoglobulin variable-region heavy chain (IgV_(H)) gene mutationstatus. High risk patients have an immature cell pattern with fewmutations in the DNA in the IgV_(H) antibody gene region whereas lowrisk patients show considerable mutations of the DNA in the antibodygene region indicating mature lymphocytes.

Since assessment of the IgV_(H) antibody DNA changes is difficult toperform, the presence of either cluster of differentiation 38 (CD38) orZ-chain-associated protein kinase-70 (ZAP-70) can be surrogate markersof high risk subtype of CLL. Their expression correlates with a moreimmature cellular state and a more rapid disease course.

In addition to the maturational state, the prognosis of patients withCLL is dependent on the genetic changes within the neoplastic cellpopulation. These genetic changes can be identified by fluorescentprobes to chromosomal parts using a technique referred to as fluorescentin situ hybridization (FISH). Four main genetic aberrations arerecognized in CLL cells that have a major impact on disease behavior.

Deletions of part of the short arm of chromosome 17 (del 17p) whichtarget the cell cycle regulating protein p53 are particularlydeleterious. Patients with this abnormality have significantly shortinterval before they require therapy and a shorter survival. Thisabnormality is found in 5-10% of patients with CLL. Deletions of thelong arm on chromosome 11 (del 11q) are also unfavorable although not tothe degree seen with del 17p. The abnormality targets the ATM gene andoccurs infrequently in CLL (5-10%). Trisomy 12, an additional chromosome12, is a relatively frequent finding occurring in 20-25% of patients andimparts an intermediate prognosis. Deletion of the long arm ofchromosome 13 (del 13q) is the most common abnormality in CLL withroughly 50% of patients with cells containing this defect. Thesepatients have the best prognosis and most will live many years, evendecades, without the need for therapy. The gene targeted by thisdeletion is a segment that likely produces small inhibitory RNAmolecules that affect expression of important death inhibiting geneproducts.

Most people are diagnosed without symptoms as the result of a routineblood test that returns a high white blood cell count. Uncommonly, CLLpresents as enlargement of the lymph nodes without a high white bloodcell count or no evidence of the disease in the blood. This is referredto as small lymphocytic lymphoma. In some individuals the disease comesto light only after the neoplastic cells overwhelm the bone marrowresulting in anemia producing tiredness or weakness.

The disease is easily diagnosed. CLL is usually first suspected by thepresence of a lymphocytosis, an increase in one type of the white bloodcell, on a complete blood count (CBC) test. This frequently is anincidental finding on a routine physician visit. Most often thelymphocyte count is greater than 4000 cells per mm³ (microliter) ofblood but can be much higher. The presence of a lymphocytosis in anelderly individual should raise strong suspicion for CLL and aconfirmatory diagnostic test, in particular flow cytometry, should beperformed unless clinically unnecessary.

The diagnosis of CLL is based on the demonstration of an abnormalpopulation of B lymphocytes in the blood, bone marrow, or tissues thatdisplay an unusual but characteristic pattern of molecules on the cellsurface. This atypical molecular pattern includes the co-expression ofcells surface markers cluster of differentiation 5 (CD5) and cluster ofdifferentiation 23 (CD23). In addition, all the CLL cells within oneindividual are clonal, that is genetically identical. In practice, thisis inferred by the detection of only one of the mutually exclusiveantibody light chains, kappa or lambda, on the entire population of theabnormal B cells. Normal B lymphocytes consist of a stew of differentantibody producing cells resulting in a mixture of both kappa and lambdaexpressing cells. The lack of the normal distribution of kappa andlambda producing B cells is one basis for demonstrating clonality, thekey element for establishing a diagnosis of any B cell malignancy (Bcell Non-Hodgkin's lymphoma).

The combination of the microscopic examination of the peripheral bloodand analysis of the lymphocytes by flow cytometry to confirm clonalityand marker molecule expression is needed to establish the diagnosis ofCLL. Both are easily accomplished on a small amount of blood. A flowcytometer is an instrument that can examine the expression of moleculeson individual cells in fluids. This requires the use of specificantibodies to marker molecules with fluorescent tags recognized by theinstrument. In CLL, the lymphocytes are genetically clonal, of the Bcell lineage (express marker molecules cluster of differentiation 19(CD19) and CD20), and characteristically express the marker moleculesCD5 and CD23. Morphologically, the cells resemble normal lymphocytesunder the microscope, although slightly smaller, and are fragile whensmeared onto a glass slide giving rise to many broken cells (smudgecells).

CLL treatment focuses on controlling the disease and its symptoms ratherthan on an outright cure. CLL is treated by chemotherapy, radiationtherapy, biological therapy, or bone marrow transplantation. Symptomsare sometimes treated surgically (splenectomy removal of enlargedspleen) or by radiation therapy (“de-bulking” swollen lymph nodes).

Initial CLL treatments vary depending on the exact diagnosis and theprogression of the disease, and even with the preference and experienceof the health care practitioner. There are dozens of agents used for CLLtherapy, and there is considerable research activity studying themindividually or in combination with each other.

While generally considered incurable, CLL progresses slowly in mostcases. Many people with CLL lead normal and active lives for manyyears—in some cases for decades. Because of its slow onset, early-stageCLL is generally not treated since it is believed that early CLLintervention does not improve survival time or quality of life. Instead,the condition is monitored over time to detect any change in the diseasepattern.

The decision to start CLL treatment is taken when the patient's clinicalsymptoms or blood counts indicate that the disease has progressed to apoint where it can affect the patient's quality of life. Determiningwhen to start treatment and by what means is often difficult; studieshave shown there is no survival advantage to treating the disease tooearly. The National Cancer Institute Working Group has issued guidelinesfor treatment, with specific markers that should be met before it isinitiated.

Although the purine analogue fludarabine was shown to give superiorresponse rates than chlorambucil as primary therapy, there is noevidence that early use of fludarabine improves overall survival, andsome clinicians prefer to reserve fludarabine for relapsed disease. Mabtherapies include alemtuzumab (directed against CD52) and rituximab(directed against CD20).

Combination chemotherapy options are effective in both newly-diagnosedand relapsed CLL. Recently, randomized trials have shown thatcombinations of purine analogues (fludarabine) with alkylating agents(cyclophosphamide) produce higher response rates and a longerprogression-free survival than single agents:

FC (fludarabine with cyclophosphamide)

FR (fludarabine with rituximab)

FCR (fludarabine, cyclophosphamide, and rituximab)

CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone)

Allogeneic bone marrow (stem cell) transplantation is rarely used as afirst-line treatment for CLL due to its risk. There is increasinginterest in the use of reduced intensity allogeneic stem celltransplantation, which offers the prospect of cure for selected patientswith a suitable donor.

Current research is comparing different forms of bone marrow transplantsto determine which patients are the best candidates and which approachis best in different situations. Younger patients that are at high riskfor dying from CLL might consider hematopoietic stem celltransplantation (HSCT). Autologous stem cell transplantation, alower-risk form of treatment using the patient's own blood cells, is notcurative. Myeloablative (bone marrow killing) forms of allogeneic stemcell transplantation, a high-risk treatment using blood cells from ahealthy donor, can be curative for some patients, but most patientscannot tolerate the treatment. An intermediate level, calledreduced-intensity conditioning allogeneic stem cell transplantation, canbe better tolerated by older or frail patients.

“Refractory” CLL is a disease that no longer responds favorably totreatment. In this case more aggressive therapies, includinglenalidomide, flavopiridol, and bone marrow (stem cell) transplantation,are considered. The monoclonal antibody, alemtuzumab (directed againstCD52), can be used in patients with refractory, bone marrow-baseddisease.

iii. Breast Cancer

Breast cancer is a cancer that starts in the breast, usually in theinner lining of the milk ducts or lobules. There are different types ofbreast cancer, with different stages (spread), aggressiveness, andgenetic makeup. With best treatment, 10-year disease-free survivalvaries from 98% to 10%. Treatment is selected from surgery, drugs(chemotherapy), and radiation. In the United States, there were 216,000cases of invasive breast cancer and 40,000 deaths in 2004. Worldwide,breast cancer is the second most common type of cancer after lung cancer(10.4% of all cancer incidence, both sexes counted) and the fifth mostcommon cause of cancer death. In 2004, breast cancer caused 519,000deaths worldwide (7% of cancer deaths; almost 1% of all deaths). Breastcancer is about 100 times as frequent among women as among men, butsurvival rates are equal in both sexes.

The first symptom, or subjective sign, of breast cancer is typically alump that feels different from the surrounding breast tissue. Accordingto the The Merck Manual, more than 80% of breast cancer cases arediscovered when the woman feels a lump. According to the American CancerSociety, the first medical sign, or objective indication of breastcancer as detected by a physician, is discovered by mammogram. Lumpsfound in lymph nodes located in the armpits can also indicate breastcancer. Indications of breast cancer other than a lump can includechanges in breast size or shape, skin dimpling, nipple inversion, orspontaneous single-nipple discharge. Pain (“mastodynia”) is anunreliable tool in determining the presence or absence of breast cancer,but can be indicative of other breast health issues.

When breast cancer cells invade the dermal lymphatics—small lymphvessels in the skin of the breast—its presentation can resemble skininflammation and thus is known as inflammatory breast cancer (IBC).Symptoms of inflammatory breast cancer include pain, swelling, warmthand redness throughout the breast, as well as an orange-peel texture tothe skin referred to as “peau d′ orange.” Another reported symptomcomplex of breast cancer is Paget's disease of the breast. This syndromepresents as eczematoid skin changes such as redness and mild flaking ofthe nipple skin. As Paget's advances, symptoms can include tingling,itching, increased sensitivity, burning, and pain. There can also bedischarge from the nipple. Approximately half of women diagnosed withPaget's also have a lump in the breast.

Occasionally, breast cancer presents as metastatic disease, that is,cancer that has spread beyond the original organ. Metastatic breastcancer will cause symptoms that depend on the location of metastasis.Common sites of metastasis include bone, liver, lung and brain.Unexplained weight loss can occasionally herald an occult breast cancer,as can symptoms of fevers or chills. Bone or joint pains can sometimesbe manifestations of metastatic breast cancer, as can jaundice orneurological symptoms. These symptoms are “non-specific,” meaning theycan also be manifestations of many other illnesses.

The primary risk factors that have been identified are sex, age,childbearing, hormones, a high-fat diet, alcohol intake, obesity, andenvironmental factors such as tobacco use, radiation and shiftwork. Noetiology is known for 95% of breast cancer cases, while approximately 5%of new breast cancers are attributable to hereditary syndromes. Inparticular, carriers of the breast cancer susceptibility genes, BRCA1and BRCA2, are at a 30-40% increased risk for breast and ovarian cancer,depending on in which portion of the protein the mutation occurs.Experts believe that 95% of inherited breast cancer can be traced to oneof these two genes. Hereditary breast cancers can take the form of asite-specific hereditary breast cancer—cancers affecting the breastonly—or breast-ovarian and other cancer syndromes. Breast cancer can beinherited both from female and male relatives.

Breast cancer subtypes are categorized on an immunohistochemical basis.Subtype definitions are generally as follows:

normal (ER+, PR+, HER2+, cytokeratin 5/6+, and HER1+)

luminal A (ER+ and/or PR+, HER2−)

luminal B (ER+ and/or PR+, HER2+)

triple-negative (ER−, PR−, HER2−)

HER2+/ER−(ER−, PR−, and HER2+)

unclassified (ER−, PR−, HER2−, cytokeratin 5/6−, and HER1−)

In the case of triple-negative breast cancer cells, the cancer's growthis not driven by estrogen or progesterone, or by growth signals comingfrom the HER2 protein. By the same token, such cancer cells do notrespond to hormonal therapy, such as tamoxifen or aromatase inhibitors,or therapies that target HER2 receptors, such as Herceptin®. About10-20% of breast cancers are found to be triple-negative. It isimportant to identify these types of cancer to that one can avoid costlyand toxic effects of therapies that are unlike to succeed, and to focuson treatments that can be used to treat triple-negative breast cancer.Like other forms of breast cancer, triple-negative breast cancer can betreated with surgery, radiation therapy, and/or chemotherapy. Oneparticularly promising approach is “neoadjuvant” therapy, where chemo-and/or radiotherapy is provided prior to surgery. Another new drugtherapy is the use of poly (ADP-ribose) polymerase, or PARP inhibitors.

While screening techniques discussed above are useful in determining thepossibility of cancer, a further testing is necessary to confirm whethera lump detected on screening is cancer, as opposed to a benignalternative such as a simple cyst. In a clinical setting, breast canceris commonly diagnosed using a “triple test” of clinical breastexamination (breast examination by a trained medical practitioner),mammography, and fine needle aspiration cytology. Both mammography andclinical breast exam, also used for screening, can indicate anapproximate likelihood that a lump is cancer, and can also identify anyother lesions. Fine Needle Aspiration and Cytology (FNAC), which can bedone in a GP's office using local anaesthetic if required, involvesattempting to extract a small portion of fluid from the lump. Clearfluid makes the lump highly unlikely to be cancerous, but bloody fluidcan be sent off for inspection under a microscope for cancerous cells.Together, these three tools can be used to diagnose breast cancer with agood degree of accuracy. Other options for biopsy include core biopsy,where a section of the breast lump is removed, and an excisional biopsy,where the entire lump is removed.

Breast cancer screening is an attempt to find cancer in otherwisehealthy individuals. The most common screening method for women is acombination of x-ray mammography and clinical breast exam. In women athigher than normal risk, such as those with a strong family history ofcancer, additional tools can include genetic testing or breast MagneticResonance Imaging.

Breast self-examination was a form of screening that was heavilyadvocated in the past, but has since fallen into disfavour since severallarge studies have shown that it does not have a survival benefit forwomen and often causes considerably anxiety. This is thought to bebecause cancers that could be detected tended to be at a relativelyadvanced stage already, whereas other methods push to identify thecancer at an earlier stage where curative treatment is more oftenpossible.

X-ray mammography uses x-rays to examine the breast for anyuncharacteristic masses or lumps. Regular mammograms is recommended inseveral countries in women over a certain age as a screening tool.

Genetic testing for breast cancer typically involves testing formutations in the BRCA genes. This is not generally a recommendedtechnique except for those at elevated risk for breast cancer.

The mainstay of breast cancer treatment is surgery when the tumor islocalized, with possible adjuvant hormonal therapy (with tamoxifen or anaromatase inhibitor), chemotherapy, and/or radiotherapy. At present, thetreatment recommendations after surgery (adjuvant therapy) follow apattern. Depending on clinical criteria (age, type of cancer, size,metastasis) patients are roughly divided to high risk and low riskcases, with each risk category following different rules for therapy.Treatment possibilities include radiation therapy, chemotherapy, hormonetherapy, and immune therapy.

Targeted cancer therapies are treatments that target specificcharacteristics of cancer cells, such as a protein that allows thecancer cells to grow in a rapid or abnormal way. Targeted therapies aregenerally less likely than chemotherapy to harm normal, healthy cells.Some targeted therapies are antibodies that work like the antibodiesmade naturally by our immune systems. These types of targeted therapiesare sometimes called immune-targeted therapies.

There are currently 3 targeted therapies doctors use to treat breastcancer. Herceptin® (trastuzumab) works against HER2-positive breastcancers by blocking the ability of the cancer cells to receive chemicalsignals that tell the cells to grow. Tykerb® (lapatinib) works againstHER2-positive breast cancers by blocking certain proteins that can causeuncontrolled cell growth. Avastin® (bevacizumab) works by blocking thegrowth of new blood vessels that cancer cells depend on to grow andfunction.

Hormonal (anti-estrogen) therapy works against hormone-receptor-positivebreast cancer in two ways: first, by lowering the amount of the hormoneestrogen in the body, and second, by blocking the action of estrogen inthe body. Most of the estrogen in women's bodies is made by the ovaries.Estrogen makes hormone-receptor-positive breast cancers grow. Soreducing the amount of estrogen or blocking its action can help shrinkhormone-receptor-positive breast cancers and reduce the risk ofhormone-receptor-positive breast cancers coming back (recurring).Hormonal therapy medicines are not effective againsthormone-receptor-negative breast cancers. There are several types ofhormonal therapy medicines, including aromatase inhibitors, selectiveestrogen receptor modulators, and estrogen receptor downregulators. Insome cases, the ovaries and fallopian tubes can be surgically removed totreat hormone-receptor-positive breast cancer or as a preventive measurefor women at very high risk of breast cancer. The ovaries also can beshut down temporarily using medication.

In planning treatment, doctors can also use PCR tests like Oncotype DXor microarray tests that predict breast cancer recurrence risk based ongene expression. In February 2007, the first breast cancer predictortest won formal approval from the Food and Drug Administration. This isa new gene test to help predict whether women with early-stage breastcancer will relapse in 5 or 10 years, this could help influence howaggressively the initial tumor is treated.

Radiation therapy is also used to help destroy cancer cells that canlinger after surgery. Radiation can reduce the risk of recurrence by50-66% when delivered in the correct dose.

E. Combination Therapy

Tumor cell resistance to single agents represents a major problem inclinical oncology. One goal of current cancer research is to find waysto improve the efficacy of existing therapies. One way is by combiningtherapies in what is known as combination therapy. In one embodiment,the present invention provides a combination therapy wherein ananti-nucleolin antibody and at least one other agent comprise acomposition for use in contacting a “target cell.” In one aspect, thecombination of anti-nucleolin antibody and at least one other agent areused in a combined amount effective to kill or inhibit proliferation ofthe cell.

In one embodiment, the method further comprises can contacting the cellswith a human anti-nucleolin antibody and one or more additional agent(s)or factor(s). In one embodiment, the method further comprises contactingthe cell with a single composition or pharmacological formulation thatincludes both agents, or by contacting the cell with two distinctcompositions or formulations, at the same time, wherein one compositionincludes the anti-nucleolin antibody and the other includes the agent.

In another embodiment, the method comprises contact by theanti-nucleolin antibody before or after contact by the other agent byintervals ranging from minutes to weeks. In embodiments where the otheragent and anti-nucleolin antibody are applied separately to the cell,one would generally ensure that a significant period of time did notexpire between the time of each delivery, such that the agent andanti-nucleolin antibody would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would contact the cell with both modalities within about 12-24 hoursof each other and, more preferably, within about 6-12 hours of eachother, with a delay time of only about 12 hours being most preferred. Insome situations, it can be desirable to extend the time period fortreatment significantly, however, where several days (2, 3, 4, 5, 6 or7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

In another embodiment, the method comprises more than one administrationof either the anti-nucleolin antibody or the other agent. Combinationsof the anti-nucleolin antibody or the other agent, include, where theanti-nucleolin antibody is “A” and the other agent is “B,” asexemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. In particular, such agents are those discussed inSection VI(C)(i)-(iii) above. In treating cancer according to theinvention, one would contact the tumor cells with an agent in additionto the anti-nucleolin antibody. This can be achieved by irradiating thelocalized tumor site with radiation or administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising a chemotherapeutic compound.

A variety of chemical compounds, also described as “chemotherapeuticagents,” function to induce DNA damage, all of which are intended to beof use in the combined treatment methods disclosed herein. In oneembodiment, the “other agents” comprise chemotherapeutic agents.Categories of chemotherapeutic agents of the invention include, but arenot limited to, alkylating agents, anthracyclines, cytoskeletaldisruptors, epothilones, inhibitors of topoisomerase II, nucleotideanalogs and precursor analogs, peptide antibiotics, platinum-basedagents, retinoids, or vinca alkaloids and derivatives. Specificchemotherapeutic agents within these groups include, but are not limitedto, actinomycin-D, all-trans retinoic acid azacitidine, adriamycinazathioprine, bleomycin, camptothecin, carboplatin, capecitabine,cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin,docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone,etoposide, fluorouracil, 5-fluorouracil (5FU), gemcitabine, hydroxyurea,hydrogen peroxide, idarubicin, imatinib, mechlorethamine,mercaptopurine, methotrexate, mitomycin C, mitoxantrone, oxaliplatin,paclitaxel, pemetrexed, teniposide, tioguanine, valrubicin, vinblastine,vincristine, vindesine, vinorelbine. The invention also encompasses theuse of a combination of one or more DNA damaging agents, whetherradiation-based or actual compounds, such as the use of X-rays withcisplatin or the use of cisplatin with etoposide.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with anti-nucleolin antibody. Agents such ascisplatin, and other DNA alkylating agents can be used. Cisplatin hasbeen widely used to treat cancer, with efficacious doses used inclinical applications of 20 mg/m² for 5 days every three weeks for atotal of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

F. Immunodetection Kits

In still further embodiments, the present invention concernsimmunodetection kits for use with the immunodetection methods describedabove. The immunodetection kits will comprise, in suitable containermeans, a human anti-nucleolin antibody, and optionally animmunodetection reagent.

In certain embodiments, the antibody can be pre-bound to a solidsupport, such as a column matrix and/or well of a microtitre plate. Theimmunodetection reagents of the kit can take any one of a variety offorms, including those detectable labels that are associated with orlinked to the given antibody. Detectable labels that are associated withor attached to a secondary binding ligand are also contemplated.Exemplary secondary ligands are those secondary antibodies that havebinding affinity for the first antibody.

Further suitable immunodetection reagents for use in the present kitsinclude the two-component reagent that comprises a secondary antibodythat has binding affinity for the first antibody, along with a thirdantibody that has binding affinity for the second antibody, the thirdantibody being linked to a detectable label. As noted above, a number ofexemplary labels are known in the art and all such labels can beemployed in connection with the present invention.

The kits can further comprise a suitably aliquoted composition of thenucleolin, whether labeled or unlabeled, as can be used to prepare astandard curve for a detection assay. The kits can containantibody-label conjugates either in fully conjugated form, in the formof intermediates, or as separate moieties to be conjugated by the userof the kit. The components of the kits can be packaged either in aqueousmedia or in lyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody can be placed, or preferably, suitably aliquoted. Thekits of the present invention will also typically include a means forcontaining the antibody, antigen, and any other reagent containers inclose confinement for commercial sale. Such containers can includeinjection or blow-molded plastic containers into which the desired vialsare retained.

VII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

In one aspect of the invention a method is provided of producing animmortalized human B-cell that secrets an antibody that binds to humannucleolin comprising: obtaining a population of IgM-positive humanB-cells; contacting said population with: Epstein-Barr virus (EBV) toimmortalize said human B-cells, and a cytokine/growth factor/signalingagent cocktail to induce IgM-to-IgG immunoglobulin isotypeclass-switching; and culturing cells under conditions supporting saidimmortalization and immunoglobulin isotype class-switching. In oneembodiment the method further comprises, selecting an immortalized humanB-cell that expresses an antibody to human nucleolin. In anotherembodiment, selecting comprises an immunoassay performed on immortalizedB-cell culture medium supernatants. In another embodiment, a cytokinecocktail comprises an agent that delivers a costimulatory signal to ahuman B-cell. In another embodiment, a cytokine cocktail comprisesanti-IgM F(ab′)₂ interleukin (IL)-2, IL-4, IL-5, IL-6, IL-9, IL-10,IL-13, INFα, BAFF, soluble CD40L. In another embodiment, a population ofIgM-positive human B-cells is obtained from peripheral blood, a tonsilsbone marrow, a spleen, a lymph node, umbilical cord blood, a liver, anapheresis procedures or a buffy coat. In another embodiment, the methodfurther comprises isolating a nucleic acid encoding an entire heavyand/or light chain from the immortalized human B-cell of step (d). Inanother embodiment, the method further comprises isolating a nucleicacid encoding a heavy and/or light chain antigen-binding region from theimmortalized human B-cell of step (d). In another embodiment, the methodfurther comprises cloning said nucleic acid into a nucleic acid encodinga framework region of a heavy and/or light chain. In another embodiment,contacting said population further comprises an EBV concentration step,a centrifugation step during infection, or both. In another embodiment,the method further comprises freezing said population of human B-cellsfollowing step (c). In another embodiment, contacting said populationwith a cytokine/growth factor/signaling agent cocktail is performed atabout 0-96 hours following step (b)(ii). In another embodiment,contacting said population with a cytokine/growth factor/signaling agentcocktail is performed at about 16-20 hours following step (b)(ii). Inanother embodiment, about 50%-99% of said population are immortalized byEBV infection. In another embodiment, about 95%-99% of said populationare immortalized by EBV infection. In another embodiment, selecting animmortalized human B-cell that expresses an antibody to human nucleolinoccurs 1-4 weeks following infection. In another embodiment, selectingan immortalized human B-cell that expresses an antibody to humannucleolin occurs 2-3 weeks following infection. In another embodiment,selecting an immortalized human B-cell that expresses an antibody tohuman nucleolin occurs after thawing stored frozen immortalized B-cells,and/or after thawing stored frozen culture medium supernatants from saidimmortalized B-cells. In another embodiment, the B-cell is antigennaïve. In another embodiment, the B-cell is antigen experienced.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody that binds to human nucleolin. In oneembodiment, the immortalized human B-cell is designated as T-5D1, V-3H11(3G5), T-2D3, T-7G7 (1H9), T-2H3, T-9F9, T-8G4 or T-P1C6.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein of SEQ ID No. 2. In one embodiment, the IgG antibody is an IgG1,IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgG antibody isan IgG1 antibody. In another embodiment, the IgG antibody comprises akappa light chain. In another embodiment, the IgG antibody comprises alambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 10% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein encoded by SEQ ID No. 1. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. The isolated antibody orfragment thereof of claim 33, wherein said antibody or fragment thereofis substantially non-immunogenic to a human. In another embodiment, theantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the antibody or fragment thereof kills at least 10%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 20% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 30%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 40% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 50%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 60% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 70%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 80% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 90%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 100% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 10%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 20% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 30%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 40% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 50%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB scrum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 60% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 70%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 80% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 90%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 100% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof binds to an RNAbinding domain of human nucleolin. In another embodiment, the antibodyor fragment thereof inactivates an RNA binding domain of humannucleolin. In another embodiment, the isolated antibody or fragmentthereof induces complement-dependent cytotoxicity to a cancer cell. Inanother embodiment, the isolated antibody or fragment thereof inducescomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the isolatedantibody or fragment thereof inhibits or kills an AML cancer cell, a CLLcancer cell or a breast cancer cell. In another embodiment, the isolatedmonoclonal antibody or fragment thereof reduces BCL-2 levels in a cancercell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein comprising SEQ ID No. 4. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the isolated antibody or fragmentthereof of claim 33, wherein said antibody or fragment thereof is amonoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 10% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an immortalized human B-cellthat expresses an IgG antibody or fragment thereof that binds to aprotein encoded by SEQ ID No. 3. In one embodiment, the IgG antibody isan IgG1, IgG2, IgG3, or IgG4 antibody. In another embodiment, the IgGantibody is an IgG1 antibody. In another embodiment, the IgG antibodycomprises a kappa light chain. In another embodiment, the IgG antibodycomprises a lambda light chain. In another embodiment, the B-cell is EBVimmortalized. In another embodiment, the antibody or fragment thereof isa monoclonal antibody or fragment thereof. In another embodiment, theantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, the antibodyor fragment thereof kills at least 20% of a population of MCF-7 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated human monoclonalantibody or fragment thereof that specifically binds to a protein of SEQID No. 4. In one embodiment, the antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 10% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 20%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 30% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 40%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 50% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 60%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 70% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 80%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof kills at least 90% of a population of MV4-11 cells,when incubated with said MCF-7 cells and human AB serum for 48 hours. Inanother embodiment, the antibody or fragment thereof kills at least 100%of a population of MV4-11 cells, when incubated with said MCF-7 cellsand human AB serum for 48 hours. In another embodiment, the antibody orfragment thereof binds to an RNA binding domain of human nucleolin. Inanother embodiment, the antibody or fragment thereof inactivates an RNAbinding domain of human nucleolin. In another embodiment, the isolatedantibody or fragment thereof induces complement-dependent cytotoxicityto a cancer cell. In another embodiment, the isolated antibody orfragment thereof induces complement-independent cytotoxicity to a cancercell. In another embodiment, the isolated antibody or fragment thereofinduces apoptosis in a cancer cell upon contact. In another embodiment,the isolated antibody or fragment thereof inhibits or kills an AMLcancer cell, a CLL cancer cell or a breast cancer cell. In anotherembodiment, the isolated monoclonal antibody or fragment thereof reducesBCL-2 levels in a cancer cell.

In another aspect the invention provides an isolated antibody orfragment thereof that specifically binds to a human nucleolin protein,wherein said antibody or fragment thereof kills at least 20% of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In one embodiment, the amino acid sequenceof said human nucleolin comprises SEQ ID No. 2. In another embodiment,the antibody or fragment thereof binds to an amino acid sequenceconsisting of amino acid residues 1 to 283 of SEQ ID No. 2. In anotherembodiment, the antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof binds to an RNA binding domain of human nucleolin. In anotherembodiment, the antibody or fragment thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the isolated antibodyor fragment thereof is linked to a diagnostic or therapeutic agent. Inanother embodiment, the isolated antibody or fragment thereof exhibitscomplement-dependent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof exhibitscomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the antibody orfragment thereof inhibits or kills an AML cancer cell, a CLL cancer cellor a breast cancer cell. In another embodiment, the isolated antibody orfragment thereof reduces BCL-2 levels in a cancer cell. In anotherembodiment, the antibody or fragment thereof is linked to a diagnosticagent. In another embodiment, the diagnostic agent is a radionuclide, afluorophore, a chemilluminescent compound, a fluorescent compound, or anenzyme. In another embodiment, the antibody or fragment thereof islinked to a therapeutic agent. In another embodiment, the therapeuticagent is a radionuclide, a toxin or a chemotherapeutic moiety.

In another aspect the invention provides an isolated antibody orfragment thereof that specifically binds to a human nucleolin protein,wherein said antibody or fragment thereof kills at least 10-100% of apopulation of MV4-11 cells (such as 10, 20, 30, 40, 50, 60, 70, 80, 90or 100% of a population of MV4-11 cells, when incubated with said MCF-7cells and human AB serum for 48 hours. In another embodiment, the aminoacid sequence of said human nucleolin comprises SEQ ID No. 2. In anotherembodiment, the antibody or fragment thereof binds to an amino acidsequence consisting of amino acid residues 1 to 283 of SEQ ID No. 2. Inanother embodiment, the antibody or fragment thereof is a monoclonalantibody or fragment thereof. In another embodiment, the antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the antibody or fragmentthereof binds to an RNA binding domain of human nucleolin. In anotherembodiment, the antibody or fragment thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the isolated antibodyor fragment thereof is linked to a diagnostic or therapeutic agent. Inanother embodiment, the isolated antibody or fragment thereof exhibitscomplement-dependent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof exhibitscomplement-independent cytotoxicity to a cancer cell. In anotherembodiment, the isolated antibody or fragment thereof induces apoptosisin a cancer cell upon contact. In another embodiment, the antibody orfragment thereof inhibits or kills an AML cancer cell, a CLL cancer cellor a breast cancer cell. In another embodiment, the isolated antibody orfragment thereof reduces BCL-2 levels in a cancer cell. In anotherembodiment, the said antibody or fragment thereof is linked to adiagnostic agent. In another embodiment, the diagnostic agent is aradionuclide, a fluorophore, a chemilluminescent compound, a fluorescentcompound, or an enzyme. In another embodiment, the antibody or fragmentthereof is linked to a therapeutic agent. In another embodiment, thetherapeutic agent is a radionuclide, a toxin or a chemotherapeuticmoiety.

In another aspect the invention provides an anti-nucleolin compositioncomprising one or more isolated antibodies or fragments thereof thatspecifically binds to a human nucleolin protein, wherein said one ormore antibodies kills at least 10-100% of a population of MCF-7 cells(such as 10, 20, 30, 40, 40, 50, 60, 70, 80, 90, or 100%), whenincubated with said MCF-7 cells and human AB serum for 48-96 (such as48, 72 or 96 hours) hours. In one embodiment, the one or more isolatedantibodies or fragments thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the one or more isolated antibodies orfragments thereof is substantially non-immunogenic to a human. Inanother embodiment, the one or more isolated antibodies or fragmentsthereof is a human antibody or fragment thereof. In another embodiment,the one or more isolated antibodies or fragments thereof binds to an RNAbinding domain of human nucleolin. In another embodiment, the one ormore isolated antibodies or fragments thereof inactivates an RNA bindingdomain of human nucleolin. In another embodiment, the amino acidsequence of said human nucleolin comprises SEQ ID No. 2. In anotherembodiment, the one or more isolated antibodies or fragments thereofbinds to SEQ ID No. 4. In another embodiment, the anti-nucleolincomposition further comprises a radionuclide, a fluorophore, achemilluminescent compound, a fluorescent compound, an enzyme, a toxinor a chemotherapeutic agent. In another embodiment, the radionuclide, afluorophore, a chemilluminescent compound, a fluorescent compound, anenzyme, a toxin or a chemotherapeutic agent is conjugated to said one ormore isolated antibodies or fragments thereof. In another embodiment,the anti-nucleolin composition comprises two or more isolated antibodiesor fragments thereof that specifically binds to said human nucleolinprotein, wherein said one or more antibodies kills at least 20% of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In another embodiment, the anti-nucleolincomposition comprises three or more isolated antibodies or fragmentsthereof that specifically binds to said human nucleolin protein, whereinsaid one or more antibodies kills at least 20% of a population of MCF-7cells, when incubated with said MCF-7 cells and human AB serum for 48hours.

In another aspect the invention provides a method of inhibiting orkilling a cell expressing nucleolin on its surface comprising contactingsaid cell with an antibody or fragment thereof that binds to humannucleolin, wherein said antibody or fragment thereof kills at least 20%of a population of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 hours. In one embodiment, the antibody or fragmentthereof is a human antibody or fragment thereof. In another embodiment,the antibody or fragment thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the antibody or fragment thereof bindsto SEQ ID No. 2. In another embodiment, the antibody or fragment thereofbinds to an amino acid sequence encoded by SEQ ID No. 1. In anotherembodiment, the antibody or fragment thereof binds to SEQ ID No. 4. Inanother embodiment, the antibody or fragment thereof binds to an aminoacid sequence encoded by SEQ ID No. 3. In another embodiment, the cellis a cancer cell. In another embodiment, the cancer cell is a lungcancer cell, a breast cancer cell, a prostate cancer cell, a coloncancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, anovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastomacell, a neuroblastoma cell, a sarcoma cell or a gastric cancer cell. Inanother embodiment, the cell is an immune cell. In another embodiment,the immune cell is a lymphocyte, dendritic cell, a peripheral bloodmonocyte, a macrophage or a glial cell. In another embodiment, theimmune cell is an activated immune cell. In another embodiment, theimmune cell is an activated B cell. In another embodiment, the immunecell is a memory B cell. In another embodiment, the immune cell is anactivated T cell. In another embodiment, the immune cell is an activatedCD4+ T cell. In another embodiment, the immune cell is an activated CD8+T cell. In another embodiment, the cell a vascular smooth muscle cell oran endothelial cell. In another embodiment, the antibody or fragmentthereof is linked to a therapeutic agent. In another embodiment, thetherapeutic agent is a radionuclide, a toxin or a chemotherapeuticagent. In another embodiment, the inhibiting or killing comprisesinducing apoptosis in said cell. In another embodiment, the cell islocated in a human subject, and said contacting comprising administeringsaid antibody or fragment thereof to said subject. In anotherembodiment, the method of further comprises contacting said cell with atleast one additional inhibitory agent or treatment. In anotherembodiment, the additional treatment comprises one or more of surgery,radiotherapy, chemotherapy, toxin therapy, immunotherapy, hormonetherapy, anti-angiogenic therapy or gene therapy or other biologicaltherapies. In another embodiment, the additional inhibitory agentcomprises one or more of radionuclides, chemotherapetic agents, toxinsimmunotherapeutics, hormones, nucleic acids or polypeptides. In anotherembodiment, the toxin is diphtheria toxin, exotoxin A chain, ricin Achain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, phytolaca americana protein, pokeweedantiviral protein, momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin, calicheamicins or tricothecenes toxin. In anotherembodiment, the chemotherapeutic agent is an alkylating agent,anthracycline, cytoskeletal disruptor, epothilone, inhibitor oftopoisomerase I, inhibitor of topoisomerase II, nucleoside or nucleotideanalog, precursor analogs, peptide antibiotic, platinum-based agentsretinoids, vinca alkaloids or derivatives thereof. In anotherembodiment, the wherein said chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of detecting a cellexpressing nucleolin on its surface comprising contacting said cell witha human antibody or fragment thereof that binds immunologically to saidnucleolin. In one embodiment, the cell is a cancer cell, an immune cell,or a vascular smooth muscle cell that expresses nucleolin on itssurface, an endothelial cell that expresses nucleolin on its surface, ora virus infected cell. In another embodiment, the cell is a precancerouscell that expresses nucleolin on its surface. In another embodiment, thecancer cell selected from the group consisting of is a lung cancer cell,a breast cancer cell, a prostate cancer cell, a colon cancer cell, apancreatic cancer cell, a renal cell carcinoma cell, an ovarian cancercell, a leukemia cell, a melanoma cell, a glioblastoma cell, aneuroblastoma cell, a sarcoma cell and a gastric cancer cell. In anotherembodiment, the immune cell is a lymphocyte, dendritic cell, aperipheral blood monocyte, a macrophage and a glial cell. In anotherembodiment, the cell is an immune cell. In another embodiment, theimmune cell is an activated immune cell. In another embodiment, theimmune cell is an activated B cell. In another embodiment, the immunecell is a memory B cell. In another embodiment, the immune cell is anactivated T cell. In another embodiment, the immune cell is an activatedCD4+ T cell. In another embodiment, the immune cell is an activated CD8+T cell. In another embodiment, the cell is a vascular smooth muscle cellor an endothelial cell. In another embodiment, the antibody or fragmentthereof is linked to a diagnostic agent. In another embodiment, thediagnostic agent is a radionuclide, a fluorophore, a chemilluminescentcompound, a fluorescent compound, a quantum dot, a nanoparticles or anenzyme. In another embodiment, the cell is located in a human subjectand contacting comprises administering said antibody or fragment thereofto said subject. In another embodiment, the cell is located in anisolated, tissue sample or cell suspension.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (such as 10, 20, 30,40, 50, 60, 70, 80, 90, or 100% of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 (such as 48,72, or 96) hours. In one embodiment, the mammal is a human. In anotherembodiment, the treating cancer in a mammal comprises treating tumorhpoxia. In another embodiment, the treating cancer in a mammal comprisesinhibiting tumor angiogenesis. In another embodiment, the anti-nucleolinantibody or fragment thereof is substantially non-immunogenic to ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is a human antibody or fragment thereof. In another embodiment,the anti-nucleolin monoclonal antibody or fragment thereof is amonoclonal antibody or fragment thereof. In another embodiment, thetoxin is diphtheria toxin, exotoxin A chain, ricin A chain, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, phytolaca americana protein, pokeweed antiviralprotein, momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, calicheamicins or tricothecenes toxin. In another embodiment,the chemotherapeutic agent is an alkylating agent, anthracycline,cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I,inhibitor of topoisomerase II, nucleoside or nucleotide analog,precursor analogs, peptide antibiotic, platinum-based agents retinoids,vinca alkaloids or derivatives thereof. In another embodiment, thechemotherapeutic agent is actinomycin-D, all-trans retinoic acidazacitidine, adriamycin azathioprine, bleomycin, camptothecin,carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide,cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin,epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil (5FU),gemcitabine, hydroxyurea, hydrogen peroxide, idarubicin, imatinib,mechlorethamine, mercaptopurine, methotrexate, mitomycin C,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,tioguanine, valrubicin, vinblastine, vincristine, vindesine, orvinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin antibody orfragment thereof, a toxin or chemotherapeutic agent and apharmaceutically acceptable carrier, wherein said antibody or fragmentthereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% of a population of MCF-7 cells, when incubated with saidMCF-7 cells and human AB serum for 48 (such as 48, 72, or 96) hours. Inone embodiment, the mammal is a human. In another embodiment, thetreating cancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin antibody orfragment thereof and a pharmaceutically acceptable carrier, and furthertreating said mammal with radiation therapy, wherein said antibody orfragment thereof kills at least 10-100% (such as 10, 20, 30, 40, 50, 60,70, 80, 90, or 100% of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 (such as 48, 72, or 96)hours. In one embodiment, the mammal is a human. In another embodiment,the treating cancer in a mammal comprises treating tumor hpoxia. Inanother embodiment, the treating cancer in a mammal comprises inhibitingtumor angiogenesis. In another embodiment, the anti-nucleolin antibodyor fragment thereof is substantially non-immunogenic to a human. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is a monoclonalantibody or fragment thereof. In another embodiment, the toxin isdiphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins, phytolaca americana protein, pokeweed antiviral protein,momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin,calicheamicins or tricothecenes toxin. In another embodiment, thechemotherapeutic agent is an alkylating agent, anthracycline,cytoskeletal disruptor, epothilone, inhibitor of topoisomerase I,inhibitor of topoisomerase II, nucleoside or nucleotide analog,precursor analogs, peptide antibiotic, platinum-based agents retinoids,vinca alkaloids or derivatives thereof. In another embodiment, thechemotherapeutic agent is actinomycin-D, all-trans retinoic acidazacitidine, adriamycin azathioprine, bleomycin, camptothecin,carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide,cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin,epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil (5FU),gemcitabine, hydroxyurea, hydrogen peroxide, idarubicin, imatinib,mechlorethamine, mercaptopurine, methotrexate, mitomycin C,mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide,tioguanine, valrubicin, vinblastine, vincristine, vindesine, orvinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In one embodiment, themammal is a human. In another embodiment, the treating cancer in amammal comprises treating tumor hypoxia. In another embodiment, thetreating cancer in a mammal comprises inhibiting tumor angiogenesis. Inanother embodiment, the anti-nucleolin antibody or fragment thereof issubstantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin monoclonalantibody or fragment thereof is a monoclonal antibody or fragmentthereof. In another embodiment, the toxin is diphtheria toxin, exotoxinA chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordii proteins, dianthin proteins, phytolaca americanaprotein, pokeweed antiviral protein, momordica charantia inhibitor,curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,restrictocin, phenomycin, enomycin, calicheamicins or tricothecenestoxin. In another embodiment, the chemotherapeutic agent is analkylating agent, anthracycline, cytoskeletal disruptor, epothilone,inhibitor of topoisomerase I, inhibitor of topoisomerase II, nucleosideor nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

A method of treating or preventing cancer in a mammal comprisingadministering to said mammal a therapeutically effective amount of aanti-nucleolin antibody or fragment thereof, a toxin or chemotherapeuticagent and a pharmaceutically acceptable carrier, wherein said antibodyor fragment thereof specifically binds to a protein of SEQ ID No. 4. Inone embodiment, the mammal is a human. In another embodiment, thetreating cancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of treating orpreventing cancer in a mammal comprising administering to said mammal atherapeutically effective amount of a anti-nucleolin antibody orfragment thereof and a pharmaceutically acceptable carrier, and furthertreating said mammal with radiation therapy, wherein said antibody orfragment thereof specifically binds to a protein of SEQ ID No. 4. In oneembodiment, the mammal is a human. In another embodiment, the treatingcancer in a mammal comprises treating tumor hpoxia. In anotherembodiment, the treating cancer in a mammal comprises inhibiting tumorangiogenesis. In another embodiment, the anti-nucleolin antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is a monoclonal antibody orfragment thereof. In another embodiment, the toxin is diphtheria toxin,exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, phytolacaamericana protein, pokeweed antiviral protein, momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, calicheamicins ortricothecenes toxin. In another embodiment, the chemotherapeutic agentis an alkylating agent, anthracycline, cytoskeletal disruptor,epothilone, inhibitor of topoisomerase I, inhibitor of topoisomerase II,nucleoside or nucleotide analog, precursor analogs, peptide antibiotic,platinum-based agents retinoids, vinca alkaloids or derivatives thereof.In another embodiment, the chemotherapeutic agent is actinomycin-D,all-trans retinoic acid azacitidine, adriamycin azathioprine, bleomycin,camptothecin, carboplatin, capecitabine, cisplatin, chlorambucil,cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine,doxorubicin, epirubicin, epothilone, etoposide, fluorouracil,5-fluorouracil (5FU), gemcitabine, hydroxyurea, hydrogen peroxide,idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate,mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine,or vinorelbine.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (such as 10, 20, 30,40, 50, 60, 70, 80, 90, or 100%) of a population of MCF-7 cells, whenincubated with said MCF-7 cells and human AB serum for 48 (such as 48,72, or 96) hours. In one embodiment, the autoimmune disease is alopeciagreata, ankylosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, asthma, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, diabetes mellitus (e.g., type 1),eosinophilic fascitis, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis,Henoch-Schonlein purpura, idiopathic pulmonary fibrosis,idiopathic/autoimmune thrombocytopenia purpura (ITP), IgA neuropathy,juvenile arthritis, lichen planus, lupus erthematosus, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1 orimmune-mediated diabetes mellitus, myasthenia gravis, pemphigus-relateddisorders (e.g., pemphigus vulgaris), myelodysplastic syndrome,pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome,Rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome,stiff-man syndrome, systemic lupus erythematosis (SLE), Sweet'ssyndrome, Still's disease, lupus erythematosus, takayasu arteritis,temporal arteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In one embodiment, theautoimmune disease is alopecia greata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, asthma,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, diabetes mellitus (e.g., type 1), eosinophilicfascites, fibromyalgia-fibromyositis, glomerulonephritis, Graves'disease, Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonleinpurpura, idiopathic pulmonary fibrosis, idiopathic/autoimmunethrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erthematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, type 1 or immune-mediated diabetesmellitus, myasthenia gravis, pemphigus-related disorders (e.g.,pemphigus vulgaris), myelodysplastic syndrome, pernicious anemia,polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still'sdisease, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating anautoimmune disease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In one embodiment, theautoimmune disease is alopecia greata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, asthma,autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia,autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmunethrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy,celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome(CFIDS), chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, diabetes mellitus (e.g., type 1), eosinophilicfascites, fibromyalgia-fibromyositis, glomerulonephritis, Graves'disease, Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonleinpurpura, idiopathic pulmonary fibrosis, idiopathic/autoimmunethrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,lichen planus, lupus erthematosus, Meniere's disease, mixed connectivetissue disease, multiple sclerosis, type 1 or immune-mediated diabetesmellitus, myasthenia gravis, pemphigus-related disorders (e.g.,pemphigus vulgaris), myelodysplastic syndrome, pernicious anemia,polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, systemic lupus erythematosis (SLE), Sweet's syndrome, Still'sdisease, lupus erythematosus, takayasu arteritis, temporalarteristis/giant cell arteritis, ulcerative colitis, uveitis,vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, andWegener's granulomatosis. Examples of inflammatory disorders include,but are not limited to, asthma, encephilitis, inflammatory boweldisease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentitatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, graft versus host disease, urticaria, orVogt-Koyanagi-Hareda syndrome. In another embodiment, the mammal is ahuman. In another embodiment, the anti-nucleolin antibody or fragmentthereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 10-100% (10, 20, 30, 40, 50,60, 70, 90, 100%) of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 (48, 72, or 96) hours. Inanother embodiment, the airway disease is asthma, chronic obstructivepulmonary disease, idiopathic pulmonary fibrosis, or inflammatorypneumonitis. In another embodiment, the mammal is a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof issubstantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In another embodiment,the airway disease is asthma, chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin antibody or fragment thereof is a monoclonal antibody orfragment thereof.

In another aspect the invention provides a method of treating an airwaydisease in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In another embodiment,the airway disease is asthma, chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis, or inflammatory pneumonitis. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is ahuman antibody or fragment thereof. In another embodiment, theanti-nucleolin antibody or fragment thereof is a monoclonal antibody orfragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof and saidantibody or fragment thereof kills at least 20% of a population of MCF-7cells, when incubated with said MCF-7 cells and human AB serum for 48hours. In another embodiment, the virally infected cell is infected withan HIV virus. In another embodiment, the mammal is a human. In anotherembodiment, the anti-nucleolin monoclonal antibody or fragment thereofis substantially non-immunogenic to a human. In another embodiment, theanti-nucleolin antibody or fragment thereof is a human antibody orfragment thereof. In another embodiment, the anti-nucleolin antibody orfragment thereof is a monoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises a human anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 2. In another embodiment,the virally infected cell is infected with an HIV virus. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating a virallyinfected cell in a mammal comprising administering to said mammal atherapeutically effective amount of an anti-nucleolin agent and apharmaceutically acceptable carrier; wherein said anti-nucleolin agentcomprises an anti-nucleolin antibody or fragment thereof thatspecifically binds to a protein of SEQ ID No. 4. In another embodiment,the virally infected cell is infected with an HIV virus. In anotherembodiment, the mammal is a human. In another embodiment, theanti-nucleolin monoclonal antibody or fragment thereof is substantiallynon-immunogenic to a human. In another embodiment, the anti-nucleolinantibody or fragment thereof is a human antibody or fragment thereof. Inanother embodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises an anti-nucleolin antibody orfragment thereof and said antibody or fragment thereof kills at least10-100% (such as 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%) of apopulation of MCF-7 cells, when incubated with said MCF-7 cells andhuman AB serum for 48 (48, 72, or 96) hours. In one embodiment, thecondition or disease in a mammal characterized by increased surfaceexpression of nucleolin is macular degeneration, diabetic retinopathy,or inflammatory disease. In another embodiment, the mammal is a human.In another embodiment, the anti-nucleolin monoclonal antibody orfragment thereof is substantially non-immunogenic to a human. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is a humanantibody or fragment thereof. In another embodiment, the anti-nucleolinantibody or fragment thereof is a monoclonal antibody or fragmentthereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises a human anti-nucleolin antibody orfragment thereof that specifically binds to a protein of SEQ ID No. 2.In one embodiment, the condition or disease in a mammal characterized byincreased surface expression of nucleolin is macular degeneration,diabetic retinopathy, or inflammatory disease. In another embodiment,the mammal is a human. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is substantially non-immunogenicto a human. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides a method of treating orpreventing a non-cancerous condition or disease in a mammalcharacterized by increased surface expression of nucleolin, comprisingadministering to said mammal a therapeutically effective amount of ananti-nucleolin agent and a pharmaceutically acceptable carrier; whereinsaid anti-nucleolin agent comprises an anti-nucleolin antibody orfragment thereof that specifically binds to a protein of SEQ ID No. 4.In one embodiment, the condition or disease in a mammal characterized byincreased surface expression of nucleolin is macular degeneration,diabetic retinopathy, or inflammatory disease. In another embodiment,the mammal is a human. In another embodiment, the anti-nucleolinmonoclonal antibody or fragment thereof is substantially non-immunogenicto a human. In another embodiment, the anti-nucleolin antibody orfragment thereof is a human antibody or fragment thereof. In anotherembodiment, the anti-nucleolin antibody or fragment thereof is amonoclonal antibody or fragment thereof.

In another aspect the invention provides an anti-nucleolin agent thatkills at least 50% of a population of MCF-7 cells, when incubated withsaid MCF-7 cells and human AB serum for 48 hours. In another embodiment,the said anti-nucleolin agent is substantially non-immunogenic to ahuman.

In another aspect the invention provides an anti-nucleolin agent thatkills more MCF-7 cells than MCF10A cells when incubated with separatepopulations of MCF-7 cells and MCF10A cells and heat inactivated serumfor 72 or 96 hours. In another embodiment, the said anti-nucleolin agentis substantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent thatkills more MCF-7 cells than MCF10A cells when incubated with separatepopulations of MCF-7 cells and MCF10A cells and human AB serum for 96hours. In another embodiment, the said anti-nucleolin agent issubstantially non-immunogenic to a human.

In another aspect the invention provides an anti-nucleolin agent thatspecifically binds to a protein of SEQ ID No. 4 and inihibits or killsone or more cancer cells that express nucleolin on their cell surface.In another embodiment, the said anti-nucleolin agent is substantiallynon-immunogenic to a human.

In another aspect the invention provides a method of determining alikelihood that a subject will develop cancer by detecting increasedcell surface nucleolin expression in one or more precancerous cells.

In another aspect the invention provides a antibody of any of theproceeding claims, wherein said antibody fragment is a Fab, Fab′,F(ab′).sub.2, or Fv fragment; diabodie; linear antibody; single-chainantibody; or a multispecific antibodt formed from an antibody fragment.

In another aspect the invention provides a method of any of theproceeding claims comprising the use of an antibody fragment, whereinsaid antibody fragment is a Fab, Fab′, F(ab′).sub.2, or Fv fragment;diabodie; linear antibody; single-chain antibody; or a multispecificantibodt formed from an antibody fragment.

Example 1 Materials & Methods

Isolation and Culture of Tonsil B Cells.

To prepare B cells from tonsils, tonsil tissue was placed inside asterile Petri dish (VWR International, cat. #25384-088) containing 20-30ml Dulbecco's phosphate buffered saline (DPBS, without CaCl₂ or MgCl₂;Gibco/Invitrogen, Grand Island, N.Y. cat. #14190144) supplemented with1× Antibiotic-Antimycotic (Gibco/Invitrogen cat. #15240-062). The tissuewas chopped and minced with scalpels to approximately 1 mm³ pieces.Additional lymphocytes were released by gentle grinding of tonsil piecesbetween the frosted glass surfaces of two sterile microscope slides (VWRcat. #12-550-34), and single cell preparation was made by strainingthrough 70 μm nylon strainer (BD Falcon, cat. #352350, BD Biosciences,Two Oak Park, Bedford, Mass.). This suspension was layered onto a Ficoll(Amersham Biosciences cat. #17-1440-03, Uppsala, Sweden) cushion (35 mlsample over 15 ml Ficoll) and resolved at 1500 G for 20 min. Theboundary layer containing mononuclear cells was extracted, washed 2×with DPBS (1300 G for 7 min), counted, and re-suspended in DPBS at 10⁸cells/ml. A highly enriched (>95%) B-cell population was obtained withthe use of StemSep Negative Selection Human B-cell Enrichment Kitantibody cocktail (cat. #14064A) and magnetic beads (cat. #19150) fromStemCell Technologies Inc., Vancouver, Canada, according tomanufacturer's instructions, with the following modifications for use ona “The Big Easy” EasySep magnet (StemCell Tech. cat. #18001). All stepswere performed in a laminar flow biohazard hood at ambient temperature.The cell suspension was placed in a sterile round bottom 14 mlpolypropylene tube (VWR cat. #60818-689), mixed with an equal volume ofthe StemSep Negative Selection Human B-cell Enrichment Kit antibodycocktail, and incubated for 10 minutes. Then, a volume of magnetic beadsuspension equal to the antibody cocktail volume was added, followed by10 minute incubation. The volume inside the tube was brought to 10 mlwith DPBS and the tube (minus the cap) was placed inside the magnet for10 minutes, at which time the contents of the tube (still inside themagnet) were gently decanted in a single pour into a second sterile 14ml tube. The original tube with non-B cells adhering to its walls wasremoved from the magnet, and the second tube was inserted for 10 minuteclean-up incubation. The enriched B-cell suspension obtained after thefirst and second negative selection steps was poured into a 15 ml Falcontube, counted, washed with DPBS (1300 G for 7 min) and resuspended in anappropriate volume of complete RPMI media for in vitro culture(generally 10⁵ to 10⁶ cells/ml) in a 37° C., 5% CO₂ tissue cultureincubator. Complete RPMI media contains RPMI 1640 (Gibco/Invitrogen cat.#11875-093) supplemented with 10% fetal bovine serum (FBS, HyClone cat.#SH30088.03, lot. #AQC23460, Logan, Utah), and 100 U/ml Penicillin, 100μg/ml Streptomycin (cat. #15140-122), 2 mM L-Glutamine (cat.#25030-081), 1 mM Sodium Pyruvate (cat. #11360-070), 10 mM HEPES (cat.#15630-080), 0.1% 2-mercaptoethanol (cat. #21985.023), and 0.1% Falk'sCloning Cocktail, which consists of 50 mM α-thioglycerol (Sigma, cat. #M6145), 20 μM bathocuproinedisulfonic acid (Sigma, cat. #B1125), 100 mMNa pyruvate (cat. #11360-070), 1M HEPES pH 7.4 (cat. #15630-080).L-glutamine, Sodium Pyruvate, Penicillin/Streptomycin and HEPES wereobtained from Gibco/Invitrogen. Alternatively, cells were resuspended incomplete RPMI media at 10⁸ cells/ml for spinfection with concentratedEpstein-Barr virus stocks to make immortalized tonsil B cell librariesas described below.

Isolation and Culture of Peripheral Blood B Cells.

To prepare B cells from peripheral blood, venous blood (up to 180 ml)was drawn into 60 ml syringes containing 1-5 ml citric acid or heparinsulfate, which prevent coagulation, diluted with equal volume of DPBS,layered onto a Ficoll cushion (35 ml of diluted sample over 15 mlFicoll) and resolved at 2000 rpm for 20 mM. Serum (from upper layer) wascollected and stored in aliquots. The boundary layer containingmononuclear cells was extracted, washed 2× with DPBS (1300 G for 7 min),counted, and re-suspended in DPBS at 10⁸ cells/ml. Highly purepopulations of B-cells were obtained with the use of StemSep NegativeHuman B-cell Enrichment Kit (StemCell Technologies Inc.) as describedabove for isolation of tonsil B-cells. Isolated B-cells were washed(1300 G for 7 min) and re-suspended at 10⁵-10⁶ cells per nil of completeRPMI media (described above), and cultured in a 37° C., 5% CO₂ tissueculture incubator. Alternatively, cells were resuspended in completeRPMI media at 10⁸ cells/ml for spinfection with concentratedEpstein-Barr virus stocks to make immortalized peripheral blood B celllibraries as described below.

EBV Stock Preparation.

To prepare infectious Epstein-Barr virus (EBV) stocks, B95-8 cells, amarmoset lymphoblastoid cell line (LCL) chronically-infected with B95-8strain EBV (Miller & Lipman, 1973), or EBfaV-GFP cells (Speck et al.,1999; described below), were cultured in complete RPMI media (describedabove) at a cell density of approximately 10⁵-2×10⁵ cells/ml, in a 37°C., 5% CO₂ tissue culture incubator.

Approximately 140 ml of cell culture (containing either B95-8 EBV orrecombinant EBfaV-GFP) was induced to enter lytic virus production phaseby treatment with phorbol myristate acetate (PMA, 10 ng/ml, Calbiochem,cat. #524400). After a four hour incubation with PMA, the PMA wasremoved from the culture supernatant and replaced with complete RPMImedia. The cells were cultured for 3 to 4 days until highly confluent,at which point cells were removed by centrifugation (1300 G for 7 min),and culture supernatant was filtered through 150 ml Nalgene 0.45 μmvacuum filter (Corning cat. #430320) to remove cell debris. Filteredsupernatant was either flash-frozen in liquid nitrogen in 1 ml-1.8 mlaliquots for storage at −80° C. in 1.5 ml Eppendorf tubes or 2 mlcryovials, or concentrated by ultrafiltration as described below.

EBV Concentration.

Viral concentration was performed by loading the filtered virussupernatant (140 ml) into two Centricon Plus-70 (100K MW cut-off) units(Millipore, Billerica, Mass.), which have a 70 ml capacity, or bysequentially loading 60 ml of viral supernatant onto a single JumboSep300K unit (Pall Corp., Ann Arbor, Mich.), which has a 60 ml capacity andwas loaded three times to concentrate up to 150 ml of filtered virusstock. All steps were performed on ice or at 4° C. according tomanufacturers' instructions. The Centricon filter units were centrifuged(2000 G) for between 15 and 45 minutes (monitored each 15 minutes),until the minimal retentate volume (approximately 0.5 ml per filtrationunit) was achieved. The filtrate was discarded, and virus-containingconcentrates were re-suspended with complete RPMI media up to a totalvolume of 14 ml (or 1/10 of the original culture supernatant volume).The JumboSep units were centrifuged at 3000 G, and yielded about 15 mlof retentate or 10× concentrated virus stocks. One ml-1.8 ml aliquots ofconcentrated virus stocks were transferred into cryovials, flash-frozenin liquid nitrogen, and transferred to −80° C. freezer for storage.

B Cell Infection by Inoculation.

B cells were resuspended at 10⁶ to 10⁷ cells/ml in complete RPMI media,and were mixed with an equal volume or up to 2 volumes of filtered EBVsupernatant, then placed in a T-25 flask and incubated for 4-6 hours ina tissue culture incubator at 37° C. and 5% CO₂. The culture volume wasthen adjusted by the addition of complete RPMI media, such that infectedcells were resuspended for cell culture at the desired concentration(generally 10⁵ to 10⁶ cells per ml), dispensed into multi-well platesand transferred to a tissue culture incubator at 37° C. and 5% CO₂.

B Cell Infection by Spinfection with Concentrated EBV Stocks.

B cells were resuspended at 10⁶ to 10⁷ cells/ml in complete RPMI media,and were mixed with an equal volume or up to two volumes of 10×concentrated EBV, or were resuspended directly in 10× concentrated EBVstocks at 2×10⁶ up to 2×10⁷ cells per ml, and placed in wells of a6-well tissue culture plate (Greiner bio-one, cat. #65760).(Concentration of EBV stocks more than 10× resulted in decreasedinfection efficiencies.) The plate was then centrifuged at 900 G for 1-2hours at ambient temperature, at which time infected cells werere-suspended in complete RPMI media, or complete RPMI media containingcytokines and signaling agents for induction of differentiation, at adesired concentration (generally 10⁵ to 10⁶ cells per ml). Infectedcells were dispensed into multi-well plates and transferred to a tissueculture incubator at 37° C. and 5% CO₂. For generation of immortalized Bcell libraries, generally ˜10⁷ cells were resuspended in 200 ml of mediacontaining cytokines and signaling agents, and plated into ten roundbottom 96-well plates (˜10⁴ cells per 200 μL per well).

Infection in the Presence of TLR Ligands.

B cells were infected with B95-8 strain EBV as described above, with theaddition of Toll-Like Receptor (TLR) ligands at the time of theinfection. The ligands were added at the following final concentrations:lipoprotein Pam3CSK4 (0.5 μg/ml), zymosan (1 μg/ml), polyinosine,polycitadylic acid (poly I:C) (25 μg/ml), lipopolysaccharide (LPS) (5μg/ml), Imiquimod (1 μg/ml), unmethylated CpG DNA (1 μg/ml). All TLRligands (from InVivoGen Inc) were generously donated by Dr. MohamedSalem (MUSC).

Evaluation of B Cell Immortalization Efficiency by Lymphoblastoid CellOutgrowth.

At 12 hours post-infection, B cells were counted and dispensed intowells of 96-well round bottom plates (Greiner cat #650180) as a 2-folddilution series, with each consecutive row of wells containing half thenumber of cells found in the previous row. The initial rows contained50,000 cells per well, and final rows in the dilution series contained24 cells per well. Cells were incubated for 9 days in a tissue cultureincubator at 37° C. and 5% CO₂, at which point lymphoblastoid celloutgrowth was visible by microscopy. Immortalization efficiency wasestimated based upon the assumption that lymphoblastoid cellproliferation resulted from EBV immortalization of at least one B cellin the well. Thus, the efficiency was calculated from rows containingwells with the lowest number of cells per well in which lymphoblastoidcell proliferation was consistently observed by microscopy, andexpressed as 1 immortalization event per number of cells originallydispensed into the well.

Flow Cytometry

analysis was performed using a Becton Dickinson FACSCalibur instrumentat the MUSC Flow Cytometry Facility, according to established methods.Infection efficiency of B cells infected with EBfaV-GFP was assessed bymeasuring fluorescence of Green Fluorescent Protein 24 h post-infection.Antibodies used to characterize human B cells were obtained from BDBiosciences and are specific for human CD20, CD19, CD23, CD27, CD30,CD38, IgD, IgM, and IgG.

Induction of B Cell Differentiation and IgG Secretion.

To determine their effect on B cell differentiation during theimmortalization process, cytokines and other signaling agents were addedto EBV infected B cells either immediately after infection, or 16 to 20hours after infection, and twice more at weekly intervals. All agentswere diluted in complete RPMI media and added to cells at the followingfinal concentrations: recombinant human interleukins (IL) IL-4, 0.2ng/ml; IL-5, 0.2 ng/ml; IL-6, 0.1 ng/ml; IL-9, 0.2 ng/ml; IL-10, 2.4ng/ml; IL-13, 1 ng/ml; recombinant human interferon-α (IFN-α2a), 2,000IU/ml; recombinant human BAFF, 1 ng/ml; recombinant human soluble CD40L,5 ng/ml; goat anti-human IgM (Fab′)₂, 1.4 μg/ml. IL-4 (cat. #200-04),IL-5 (cat. #200-05), IL-6 (cat. #200-06), IL-9 (cat. #200-09), IL-10(cat. #200-10), IL-13 (cat. #200-13), CD40L (cat. #310-02) and BAFF(cat. #310-13) were obtained from PeproTech (Rocky Hill, N.J.). IFN-α2a(Roferon^(R)-A) was from Roche Pharmaceuticals, and goat anti-human IgM(Fab′)₂ (cat. #109-006-129) was from Jackson Immune ResearchLaboratories Inc.

Creation of Immortalized B Cell Repertoires Used in Nucleolin BindingStudies.

Tonsil or peripheral blood B cells were infected by spinfection with 10×concentrated B95-8 virus as described above. Immediately followingspinfection, cells were resuspended in complete RPMI media to whichCD40L (5 ng/mL), BAFF (1 ng/ml), and goat anti-human IgM (Fab′)₂ (1.4μg/ml) (for twelve samples) were added. Generally tonsil librariesconsisted of ˜10⁷ infected B cells that were resuspended in 200 ml ofmedia containing the cytokines and signaling agents cocktail, and platedinto ten round bottom 96-well plates (˜10⁴ cells per 200 μL per well).Peripheral blood libraries were resuspended at 10⁶ B cells per 20 mlmedia plus cocktail, and were plated into round bottom 96-well plates at˜10⁴ cells per 200 per well.

Measurement of Human Immunoglobulin IgM and IgG Production by ELISA.

Culture supernatants were collected at various time points beginning 5days after infection and stored frozen at −20° C. until quantitativeassay by capture ELISA for IgM and IgG. Costar EIA/RIA 96-well plateswere coated with goat anti-human IgG UNLB or anti-human IgM UNLB(Southern Biotech) at 2 μg/ml in 0.05M carbonate-bicarbonate buffer, pH9.6, using 100 μl/well. Covered plates are incubated overnight at 4° C.Next day, plates are washed 4 times with 1×PBS/0.05% Tween-20 (200μl/well), and blocked with 1×PBS/1% BSA (300 μl/well) for 1 hr at roomtemperature with shaking at 450 rpm. Plates are then washed 2 times with1×PBS/0.05% Tween-20 (3500wen), and samples and standards (human IgG andhuman IgM standards (Sigma) are diluted in 1×PBS/1% BSA and applied toplates (100 μl/well). A 10-point standard curve with 2-fold dilutionsranging from 3.9 ng/mL to 1000 ng/mL, and media only is created. Platesare then incubated for 1 hour at room temperature, with shaking at 450rpm. After washing, plates are washed 4 times with 200 μl/well of1×PBS/0.05% Tween-20, and detection antibody is then added (goatanti-human IgG or IgM AP-conjugate (Southern Biotech #2040-04 or2020-04) diluted 1:4000 in 1×PBS/1% BSA (100 μl/well). Plates areincubated for 1 hour at room temperature, with shaking at 450 rpm. Afterwashing, AP conversion of colorimetric substrate p-nitrophenyl phosphatedisodium salt (PNPP, Peirce cat #37620) was detected by measuringabsorbance at OD₄₀₅ using a Multiskan Spectrum plate reader(ThermoLabsystems). Levels of human immunoglobulin in culturesupernatant samples were calculated following standard curve calibrationof purified human IgG and IgM standards using MultiSkan software.

Nucleolin Binding ELISA.

Screening by ELISA for recombinant nucleolin binding was performed bycoating plates overnight at 4° C. with recombinant nucleolin (0.5-2μg/ml) in 1× Dulbeccos PBS Ca/Mg-free. Next day, plates are washed 3times with 1×PBS/0.05% Tween-20 (300 μL per well) and blocked with 1×SuperBlock protein (Thermo Scientific #37545) in 2.5 mM Tris, 0.15 MNaCl, pH 7.4, for 1 h at room temperature with shaking at 450 rpm. Afterwashing, primary antibody is added, consisting of either control mouseanti-nucleolin MS-3 MAb (Santa Cruz Biotechnology, Inc. #sc-8031)diluted 1:100 in blocking buffer, or B cell culture supernatantscontaining anti-nucleolin antibodies (neat, 100 μL/well). After 2 hincubation at room temperature with shaking at 450 rpm, plates arewashed and detection antibody is added, consisting of either goatanti-human IgG-HRP MAb (Southern Biotech, #2040-05) used at 1:5000dilution in blocking buffer for culture supernatants, or for MS3 goatanti-mouse IgG-HRP MAb (Santa Cruz Biotechnology, Inc. #sc-2055) used at1:2000 dilution in blocking buffer for detection of control mouse MS3MAb. Plates were developed using TMB+ chromagen (Dako, Denmark S1599)used at 100 μL/well, and quantified by spectrophotometry at absorbanceOD₄₅₀.

Production and Isolation of Recombinant Human Nucleolin Protein.

E. coli was transformed with a recombinant pET21 a plasmid carrying atruncated nucleolin gene encoding residues 284-707 and six histidines[pET Δ1-283 Nuc-(His)₆] (Yang et al., 2002), and lysed by sonication.Recombinant human nucleolin was produced and purified as describedpreviously (Sengupta et al., 2004). Briefly, E. coli were cultured untilA600=0.6, then induced overnight with 0.4 mMisopropyl-1-thio-β-D-galactopyranoside at 30° C. After induction, cellpellets were resuspended in buffer A (20 mM sodium phosphate buffer, pH7.4, 500 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride)and lysed by sonication. The lysate was centrifuged at 12,000 g for 30min. The supernatant was loaded onto a 1.7 ml metal chelate (POROS MC-M)column (BioCAD SPRINT perfusion chromatography system) equilibrated andwashed with buffer A, then eluted with a linear gradient of 0-200 mMimidazole in buffer C. The imidazole was removed by dialysis, and theprotein concentrated in a Microcon concentrator. The proteinconcentration was measured by Bradford assay, and protein purity wasassessed by SDS-PAGE analysis.

Isolation of Endogenous Nucleolin from MV4-11 Cells.

A human nucleolin affinity column was created by coupling mouseanti-human nucleolin MAb MS-3 (Santa Cruz) to Ultralink Biosupportmedium (Pierce) according to the manufacturer's instructions. Briefly, 1mg of MS-3 MAb was dialyzed against 0.1 M carbonate buffer, pH 9.0, andsodium citrate was added to a final concentration of 0.6 M. Dry beads(0.25 g) were added and mixed for 2 h at RT, poured into a column andwashed with PBS. The eluate and washings were collected to determine thecoupling efficiency, which is typically ˜94%. Six ml of 3 Methanolamine, pH 9.0, was added to the column to block residual bindingsites, and then the column was closed at both ends and mixedend-over-end for 2.5 h at RT. The column was then washed sequentiallywith PBS, 1 M NaCl, then PBS, and the MV4-11 cell extract was loaded. Tomake the cell extract, MV4-11 cells were washed with PBS, pelleted andresuspended in lysis buffer containing protease and phosphataseinhibitors and incubated for 15 min on ice. The lysate was centrifugedat 10,000 g for 20 min and the supernatant loaded onto the MS-3MAb-affinity column. The column was washed with 20 volumes ofTris-buffer (pH 7.5) and the bound proteins eluted with antigen/antibodyelution buffer (Pierce). One ml fractions were collected and desaltedusing PD10 columns. The protein fraction was concentrated and analyzedby SDS-page followed by silver staining and western blotting with MS-3anti-nucleolin MAb.

Clone 5D1.

Initial Screening and Primary Subcloning.

Tonsil sample was received processed into 5×96-well plates: repertoire(T031009). Plate pools were screened for plate pools and well pools(well pools for P031109 and T031009 combined). Putative positive wells(T031009-5D1 and -5G5, P030909-3D1 and -3G5) were expanded from 1 wellinto 3 wells each. ELISA confirmation was conducted, identifyingreactive wells (T031009-5D1 and P031109-3G5). The wells were subclonedinto 1 plate of 1000 cells/well and 2 plates of 100 cells/well.

Secondary Subcloning.

The 3 subclone plates (1×1000 cells/well and 2×100 cells/well) werescreened as a single plate pool without reactivity, and rescreened.Reactive wells were confirmed. T031009-5D1 wells 1E7 and 1G7 had thehighest reactivity and were each subcloned into 60 wells on individual96-well plates.

Tertiary Subcloning.

The 2 subclone plates (T031009-5D1-1E7 and T031009-5D1-1G7) werescreened as a single plate pool three times, without reactivity; howeverthe positive control mouse monoclonal antibody (MS-3) was alsonon-reactive indicating that the nucleolin antigen had degraded, andunfortunately there was none remaining for further screening. Plateswere split twice with the excess cells separately pooled and outgrownand aliquots were frozen. The plate pools and outgrown replicate poolswere retested against new stocks of non-degraded nucleolin. Wells 1C11and 1G2 (both from T031009-5D1-1G7) were weakly reactive, and selectedfor subcloning.

Quaternary Subcloning.

The 2 subclone plates were screened 2 weeks later as a plate poolwithout reactivity, and were rescreened one week later showing weakreactivity in multiple wells. Individual wells were screened, andT031009-5D1-1G7 wells 1F10 and 1G2 had the highest reactivity and wereeach subcloned into 60 wells on individual 96-well plates.

5^(th) Round Subcloning.

The subclone plates were screened 2 weeks later as single column poolswith very weak reactivity in 1 and 2 wells each, and were rescreened aweek later, and the full plate pool was rescreened 2 days after thatwith individual wells confirmed 2 days later. T031009-5D1-1G7-1F10 well1D10 had the highest reactivity and was subcloned into 60 wells on asingle 96-well plate.

6^(th) Round Subcloning.

The subclone plate was screened 16 days later as an individual columnpool with 3 reactive wells. Screening of the full plate was done 4 dayslater and T031009-5D1-1G7-1F10-1D10 well 1B4 had the highest reactivityand was subcloned 2 days later into 60 wells on a single 96-well plate.

7^(th) Round Subcloning.

The subclone plate was screened 17 days later as an individual columnpool without reactivity, and rescreened 10 days later with moderatereactivity in 4 wells and weak reactivity in the other 2 wells.Screening of the entire plate was done 2 days later andT031009-5D1-1G7-1F10-1D10-1B4 well 1C7 had the highest reactivity andwas subcloned into 60 wells on a single 96-well plate 2 days later. Theremainder of the reactive wells was pooled, expanded and frozen at −80°C. (T031009-5D1-1G7-1F10-1D10-1B4).

8^(th) Round Subcloning.

Three weeks later, the subclone plate was screened as an individualcolumn pool with all 6 wells moderately reactive. Screening of theentire plate was done with many strongly or moderately reactive wells.T031009-5D1-1G7-1F10-1D10-1B4-1C7 well 1G10 had the highest reactivityand was subcloned into 60 wells on a single 96-well plate. The remainderof the reactive wells was pooled. Eleven days later, the subclonedplates showed evidence of fungal contamination and were discarded.Reactive cells from the pooled remainder(T031009-5D1-1G7-1F10-1D10-1B4-1C7) were expanded and 4 days latersubcloned into ten 96-well plates. Two weeks later these subclone plateswere screened, and T031009-5D1-1G7-1F10-1D10-1B4-1C7 wells 8B9 and 8E10had the highest nucleolin reactivity and were subcloned the next dayinto 60 wells each on single 96-well plates. Nine days later, thesubcloned plates again showed evidence of fungal contamination and werediscarded. Frozen pooled T031009-5D1-107-1F10-1D10-1B4 cells were thawedand expanded, and ten days later, subcloned into five 96-well plates.Two weeks later, the plates were screened for nucleolin reactivity, andT031009-5D1-107-1F10-1D10-1B4 wells 2E3 and 4C9 were confirmed positiveon the following day, and were subcloned into 60 wells each on single96-well plates. Ten days later, the plates were screened for nucleolinreactivity, and T031009-5D1-1G7-1F10-1D10-1B4-2E3 wells 2D10 and 2G5 hadthe highest reactivity, and were subcloned the next day into 60 wellseach on single 96-well plates.

9^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and T031009-5D1-1G7-1F10-1D10-1B4-2E3-2D10 well 1F7 had thehighest nucleolin reactivity, and was subcloned the next day by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweenweeks 4 and 6. Three wells (6G10, 2F8, 6B3) had reactivity. CloneT031009-5D1-1G7-1F10-1D10-1B4-2E3-2D10-1F7-6B3 was expanded forfunctional antibody testing because of its superior growth properties.

Clone 3H11.

Initial Screening and Primary Subcloning.

Volunteer peripheral blood sample received, processed into 5×96-wellplates: repertoire (P031109). Screened for plate pools and well pools(well pools for P031109 and T031009 combined). Putative positive wells(T031009-5D1 and -5G5, P031109-3D1 and -3G5) were expanded from 1 wellinto 3 wells each and P031109-3G5 was identified as the reactive well.It was subcloned into 1 plate of 500 cells/well and 2 plates of 50cells/well.

Secondary Subcloning.

The 3 subclone plates (1×500 cell/well and 2×50 cell/well) were screenedfor reactivity as a single plate pool without reactivity. Plate pool wasrescreened. Reactive wells were confirmed and P031109-3G5 wells 1D9 and1D11 had the highest reactivity and each were subcloned into 60 wells onsingle 96-well plates.

Tertiary Subcloning, Loss and Recovery.

The 2 subclone plates (P031109-3G5-1D9 and P031109-3G5-1D11) werescreened as a single plate pool twice without reactivity; however, thepositive control mouse monoclonal antibody (MS-3) was also non-reactivesuggesting that the nucleolin antigen had degraded, and unfortunatelythere was none remaining for further screening. Plates were split twicewith the excess cells separately pooled and outgrown, and aliquots werefrozen. The plate pools and outgrown replicate pools were tested againstnew stocks of non-degraded nucleolin. The original subclone plates lostreactivity while the outgrown replicate supernatants tested positive.These cells expanded from P031109-3G5-1D9 were subcloned into 10×96-wellplates and cultured.

Quaternary Subcloning.

The 10 subclone plates (P031109-3G5-1D9) were screened as a single platepool, which was poorly reactive. The plate pool supernatant wasre-screened with weak reactivity. Individual wells were tested.P031109-305-1D9 well 3H11 had the highest reactivity and was subclonedinto 60 wells on a single 96-well plate.

5^(th) Round Subcloning.

The subclone plate was screened as a single column pool without anyreactivity, and rescreened with 2 positive wells. Screening of the fullplate was done. P031109-3G5-1D9-3H11 well 1F8 had the highest reactivityand was subcloned into 60 wells on a single 96-well plate.

6^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with 4wells indicating reactivity. Screening of the full plate was done andP031109-3G5-1D9-3H11-1F8 well 1C7 had the highest reactivity and wassubcloned into 60 wells on a single 96-well plate.

7^(th) Round Subcloning.

The subclone plate was screened as a column pool with weak reactivity in2 wells. Rescreening was done with moderate reactivity in 5 wells.Screening of the entire plate was done and P031109-305-1D9-3H11-1F8-1C7well 1F8 had the highest reactivity and was subcloned into 60 wells on asingle 96-well plate. The remainder of the reactive wells was pooled,expanded and frozen at −80° C. (P031109-3G5-1D9-3H11-1F8-1C7).

8^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with 2highly reactive and 4 moderately reactive wells. Screening of the entireplate was done with multiple strongly reactive wells.P031109-3G5-1D9-3H11-1F8-1C7-1F8 well 1C3 had the highest reactivity andwas subcloned into 60 wells on a single 96-well plate. The remainder ofthe reactive wells was pooled. Eleven days later, the subcloned platesshowed evidence of fungal contamination and were discarded. Reactivecells from the pooled remainder (P031109-3G5-1D9-3H11-1F8-1C7-1F8) wereexpanded and 7 days later subcloned into ten 96-well plates. Two weekslater these subclone plates were screened, andP031109-3G5-1D9-3H11-1F8-1C7-1F8 wells 8B9 and 8E10 had the highestnucleolin reactivity and were subcloned the next day into 60 wells eachon single 96-well plates. Nine days later, the subcloned plates againshowed evidence of fungal contamination and were discarded. Frozenpooled P031109-3G5-1D9-3H1′-1F8-1C7 cells were thawed and expanded, andten days later, subcloned into five 96-well plates. Two weeks later, theplates were screened for nucleolin reactivity, andP031109-3G5-1D9-3H11-1F8-1C7 wells 2D2 and 2G9 were confirmed positiveon the following day, and were subcloned into 60 wells each on single96-well plates. Ten days later, the plates were screened for nucleolinreactivity, and P031109-3G5-1D9-3H11-1F8-1C₇₋₂G9 wells 2E4 and 2G3 hadthe highest reactivity, and were subcloned the next day into 60 wellseach on single 96-well plates.

9^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and P031109-3G5-1D9-3H11-1F8-1C₇₋₂G9-2E4 well 1B9 had thehighest nucleolin reactivity, and was subcloned the next day by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweenweeks 4 and 6. Four wells (8D2, 3F3, 2C6, 9B7) had reactivity. CloneP031109-3G5-1D9-3H11-1F8-1C₇₋₂G9-2E4-1B9-2C6 was expanded for functionalantibody testing because of its superior growth properties.

Clone 2D3.

Initial Screening and Primary Subcloning.

Tonsil sample received and processed into 10×96-well plates: repertoire(T031609A). Eleven days later these were screened for plate pools andwell pools. ELISA confirmation was conducted 3 days later, and reactivewell (T031609A-2D3) was identified and subcloned into 1 plate of 1000cells/well and 2 plates of 100 cells/well 2 days later.

Secondary Subcloning.

The 3 subclone plates (1×1000 cells/well and 2×100 cells/well) werescreened as a single plate pool without reactivity, and rescreened again1 week later. Reactive wells were confirmed. T031609A-2D3 wells 1C7 and1E3 had the highest reactivity and were each subcloned into 60 wells onindividual 96-well plates.

Tertiary Subcloning.

The 2 subcloned plates (T031609A-2D3-1C7 and T031609A-2D3-1E3) werescreened as a single plate pool, and repeated without reactivity;however, the positive control mouse monoclonal antibody (MS-3) was alsonon-reactive, indicating that the nucleolin antigen was degraded, andunfortunately there was none remaining for further screening. Plateswere split twice with the excess cells separately pooled and outgrownand aliquots frozen. The plates and outgrown replicate pools were testedagainst new stocks of non-degraded nucleolin. Reactive well 1F4 (fromT031609A-2D3-1C7) was selected and subcloned.

Quaternary Subcloning.

The subclone supernatants (T031609A-2D3-1C7-1F4) were screened withoutreactivity, and rescreened with weak reactivity. Wells 1B11 and 1F3 hadthe highest reactivity and were selected for subcloning. Each well wassubcloned into 60 wells on individual 96-well plates.

5^(th) Round Subcloning.

The subclone plates were screened as single column pools withoutreactivity, and rescreened, with plate pools rescreened, andconfirmation done. Well 1C9 from plate T031609A-2D3-1C7-1F4-1B11 had thehighest reactivity and was subcloned into 60 wells on a single 96-wellplate.

6^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with 3wells indicating reactivity. Screening of the full plate was done.

T031609A-2D3-1C7-1F4-1B11-1C9 well 1G6 had the highest reactivity andwas subcloned into 60 wells on a single 96-well plate.

7^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with weakreactivity in 1 well. Rescreening was done with moderate reactivity intwo wells. Screening of the entire plate was done andT031609A-2D3-1C7-1F4-1B11-1C9-1G6 well 1E10 had the highest reactivityand was subcloned into 60 wells on a single 96-well plate. The remainderof the reactive wells was pooled, expanded and frozen at −80° C.(T031609A-2D3-1C7-1F4-1B11-1C9-1G6).

8^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with 4reactive wells. Screening of the entire plate was done with moderatereactivity. T031609A-2D3-1C7-1F4-1B11-1C9-1G6-1E10 well 1F3 had thehighest reactivity and was subcloned into 60 wells on a single 96-wellplate. The remainder of the reactive wells was pooled. Eleven dayslater, the subcloned plates showed evidence of fungal contamination andwere discarded. Reactive cells from the pooled remainder(T031609A-2D3-1C7-1F4-1B11-1C9-1G6-1E10) were expanded and 7 days latersubcloned into ten 96-well plates. Two weeks later these subclone plateswere screened, and T031609A-2D3-1C7-1F4-1B11-1C9-1G6-1E10 wells 10F7 and10G7 had the highest nucleolin reactivity and were subcloned the nextday into 60 wells each on single 96-well plates. Nine days later, thesubcloned plates again showed evidence of fungal contamination and werediscarded. Frozen pooled T031609A-2D3-1C7-1F4-1B11-1C9-1G6 cells werethawed and expanded, and ten days later, subcloned into five 96-wellplates. Two weeks later, the plates were screened for nucleolinreactivity, and T031609A-2D3-1C7-1F4-1B11-1C9-1G6 wells 1B6 and 5F4 wereconfirmed positive on the following day, and were subcloned into 60wells each on single 96-well plates. Ten days later, the plates werescreened for nucleolin reactivity, andT031609A-2D3-1C7-1F4-1B11-1C9-1G6-5F4 wells 2F10 and 2G6 had the highestreactivity, and were subcloned the next day into 60 wells each on single96-well plates.

9^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and T031609A-2D3-1C7-1F4-1B11-1C9-1G6-5F4-2F10 well 1D8 hadthe highest nucleolin reactivity, and was subcloned the next day bylimiting dilution cloning into ten 96-well plates. Clonal coloniesappeared slowly over time, and were screened for nucleolin reactivitybetween weeks 4 and 6. Three wells (9D7, 2A12, 3E4) had reactivity.Clone T031609A-2D3-1C7-1F4-1B11-1C9-1G6-5F4-2F10-1D8-2A12 was expandedfor functional antibody testing because of its superior growthproperties.

Clone 7G7.

Initial Screening and Primary Subcloning.

Tonsil sample received and processed into 10×96-well plates: repertoire(T031609B). These were screened for plate pools and well pools. ELISAconfirmation was conducted, identifying reactive well T031609B-1H9,which was subcloned into 3 plates (1 plate of 1000 cells/well and 2plates of 100 cells/well).

Secondary Subcloning.

The 3 subclone plates (1×1000 cell/well and 2×100 cell/well) werescreened as a single plate pool without reactivity, and rescreenedlater. Reactive wells were confirmed. T031609B-1H9 wells 1G2 and 1G9 hadthe highest reactivity and were each subcloned into 60 wells onindividual 96-well plates.

Tertiary Subclones Loss and Recovery.

The 2 subclone plates (T031609B-1H9-1G2 and T031609B-1′-19-1G9) werescreened as a single plate pool three times without reactivity; however,the positive control mouse monoclonal antibody (MS-3) was alsonon-reactive indicating that the nucleolin antigen had degraded, andunfortunately there was none remaining for further screening. Plateswere split twice with the excess cells separately pooled and outgrownand aliquots were frozen. The plate pools and outgrown replicate poolswere tested against new stocks of non-degraded nucleolin, but werenon-reactive. Frozen aliquots from T031609B-1H9-1G2 were thawed on Jun.3, 2009, and plated into 10×96-well plates for reculture.

Quaternary Subcloning.

The 10 subclone plates (T031609B-1H9-1G2) were screened as a column poolwithout reactivity. Supernatants from the 10 pooled plates wererescreened with weak reactivity. Individual wells were tested.T031609B-1H9-1G2 well 7G7 had the highest reactivity and was subclonedinto 60 wells on a single 96-well plate.

5^(th) Round Subcloning.

The subclone plate was screened as a single column pool without anyreactivity, and rescreened later with 1 reactive well. The full platepool was rescreened, and T031609B-1H9-1G2-7G7 well 1B9 had the highestreactivity and was subcloned into 60 wells on a single 96-well plate.

6th Round Subcloning.

The subclone plate was screened as an individual column pool with 2reactive wells. Screening of the full plate was done andT031609B-1H9-1G2-7G7-1B9 well 1D6 had the highest reactivity and wassubcloned into 60 wells on a single 96-well plate.

7th Round Subcloning.

The subclone plate was screened as a column pool with weak reactivity in4 wells, and rescreened on with moderate reactivity in 4 wells.Screening of the entire plate was done and T031609B-1H9-1G2-7G7-1B9-1D6well 1E3 had the highest reactivity and was subcloned into 60 wells on asingle 96-well plate. The remainder of the reactive wells was pooled,expanded and frozen at −80° C. (T031609B-1H9-1G2-7G7-189-1D6).

8^(th) Round Subcloning.

The subclone plate was screened as an individual column pool with 5moderately reactive wells. Screening of the entire plate was done withmultiple moderately reactivity. T031609B-1H9-1G2-707-1B9-1D6-1E3 well1D9 had the highest reactivity and was subcloned into 60 wells on asingle 96-well plate. The remainder of the reactive wells was pooled.Eleven days later, the subcloned plates showed evidence of fungalcontamination and were discarded. Reactive cells from the pooledremainder (T031609B-1H9-1G2-7G7-1B9-1D6-1E3) were expanded and 7 dayslater subcloned into ten 96-well plates. Two weeks later these subcloneplates were screened, and T031609B-1H9-1G2-7G7-1B9-1D6-1E3 wells 3G10and 3G11 had the highest nucleolin reactivity and were subcloned thenext day into 60 wells each on single 96-well plates. Nine days later,the subcloned plates again showed evidence of fungal contamination andwere discarded. Frozen pooled T031609B-1H9-1G2-7G7-1B9-1D6 cells werethawed and expanded, and ten days later, subcloned into five 96-wellplates. Two weeks later, the plates were screened for nucleolinreactivity, and T031609B-1H9-1G2-7G7-1B9-1D6 wells 3D9 and 4B7 wereconfirmed positive on the following day, and were subcloned into 60wells each on single 96-well plates. Ten days later, the plates werescreened for nucleolin reactivity, and T031609B-1H9-1G2-7G7-1B9-1D6-4B7wells 2C10 and 2F11 had the highest reactivity, and were subcloned thenext day into 60 wells each on single 96-well plates.

9^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and T031609B-1H9-1G2-7G7-1B9-1D6-4B7-2F11 well 2E6 had thehighest nucleolin reactivity, and was subcloned the next day by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweenweeks 4 and 6. Four wells (2B9, 7F2, 1C4, 10G11) had reactivity. CloneT031609B-1H9-1G2-7G7-1B9-1D6-4B7-2F11-2E6-7F2 was expanded forfunctional antibody testing because of its superior growth properties.

Clone 2H3.

Initial Screening and Primary Subcloning.

2 tonsil samples were received, pooled and processed into a singlerepertoire consisting of 10×96-well plates (T060809). This was screened10 days later for plate pools and well pools. ELISA confirmationscreening was conducted the next day and reactive well T060809-2H3 wasidentified and subcloned 3 days later into 60 wells of a single plate.

Secondary Subcloning.

The subclone plate was screened as a single column pool withoutsubstantial reactivity, and column pools were rescreened, with the fullplate rescreened. T060809-2H3 wells 1B8 and 1E7 had the highestreactivity and were each subcloned into 60 wells on individual 96-wellplates.

Tertiary Subcloning.

The subclone plates were screened as individual column pools withmultiple wells indicating reactivity. Screening of full plates was done.Reactive wells T060809-2H3-1B8 1C10 and T060809-2H3-1E7 1F5 had thehighest reactivity and were each subcloned into 60 wells on individual96-well plates.

Quaternary Subcloning.

The subclone plates were screened as individual column pools withmultiple reactive wells for T060809-2H3-1B8-1C10. Screening of the fullplate was done and well 1F9 had the highest reactivity and was subclonedinto 60 wells on a single 96-well plate. The remainder of the reactivewells was pooled, expanded and frozen at −80° C. (T060809-2H3-1B8-1C10).

5^(th) Round Subcloning.

The subclone plate was screened as an individual column pool withoutreactivity, and rescreened later, while the entire plate was done withmoderate reactivity in many wells. T060809-2H3-1B8-1C10-1F9 well 1G3 hadthe highest reactivity was subcloned into 60 wells on a single 96-wellplate. The remainder of the reactive wells was pooled. Eleven dayslater, the subcloned plates showed evidence of fungal contamination andwere discarded. Reactive cells from the pooled remainder(T060809-2H3-1B8-1C10-1F9) were expanded and 7 days later subcloned intoten 96-well plates. Two weeks later these subclone plates were screened,and T060809-2H3-1B8-1C10-1F9 wells 2D8 and 3F9 had the highest nucleolinreactivity and were subcloned the next day into 60 wells each on single96-well plates. Nine days later, the subcloned plates again showedevidence of fungal contamination and were discarded. Frozen pooledT060809-2H3-1B8-1C10 cells were thawed and expanded, and ten days later,subcloned into five 96-well plates. Two weeks later, the plates werescreened for nucleolin reactivity, and T060809-2H3-1B8-1C10 wells 1B7and 1D6 were confirmed positive on the following day, and were subclonedinto 60 wells each on single 96-well plates. Ten days later, the plateswere screened for nucleolin reactivity, and T060809-2H3-1B8-1C10-1D6wells 2D11 and 2E9 had the highest reactivity, and were subcloned thenext day into 60 wells each on single 96-well plates.

6^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and T060809-2H3-1B8-1C10-1D6-2D11 well 2C7 had the highestnucleolin reactivity, and was subcloned the next day by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweenweeks 4 and 6. Four wells (3D8, 10C3, 7G9, 1H4) had reactivity. CloneT060809-2H3-1B8-1C10-1D6-2D11-2C7-1H4 was expanded for functionalantibody testing because of its superior growth properties.

Clone 9F9.

Initial Screening and Primary Subcloning.

2 tonsil samples were received, pooled and processed into a singlerepertoire consisting of 10×96-well plates (T061509). These werescreened for plate pools and well pools 10 days later. ELISAconfirmation was conducted the next day, identifying reactive wellT061509-9F9, which was subcloned into 60 wells on 1 plate the followingday.

Secondary Subcloning.

The subclone plate was screened as a single column pool without anyreactivity, and was rescreened later. Then the full plate wasrescreened. T061509-9F9 well 1D11 had the highest reactivity and wassubcloned into 60 wells on a single 96-well plate.

Tertiary Subcloning.

The subclone plate was screened as an individual column pool with onereactive well. Screening of the full plate was done later andT061509-9F9-1D11 well 1D10 had the highest reactivity and was subclonedinto 60 wells on a single 96-well plate.

Quaternary Subcloning.

The subclone plates were screened as individual column pools withoutreactivity, and rescreened again without reactivity. Screening of theentire plate was done later with moderate reactivity.T061509-9F9-1D11-1D10 well 1F9 had the highest reactivity and wassubcloned into 60 wells on a single 96-well plate. The remainder of thereactive wells was pooled, expanded and frozen at −80° C.(T061509-9F9-1D11-1D10).

5th Round Subcloning.

The subclone plates were screened as individual column pools withmultiple reactive wells. Screening of the entire plate was done withmoderate reactivity. T061509-9F9-1D11-1D10-1F9 well 1B6 had the highestreactivity and was subcloned into 60 wells on a single 96-well plate.The remainder of the reactive wells was pooled. Eleven days later, thesubcloned plates showed evidence of fungal contamination and werediscarded. Reactive cells from the pooled remainder(T061509-9F9-1D11-1D10-1F9) were expanded and 7 days later subclonedinto ten 96-well plates. Two weeks later these subclone plates werescreened, and T061509-9F9-1D11-1D10-1F9 wells 6B9 and 9D3 had thehighest nucleolin reactivity and were subcloned the next day into 60wells each on single 96-well plates. Nine days later, the subclonedplates again showed evidence of fungal contamination and were discarded.Frozen pooled T061509-9F9-1D11-1D10 cells were thawed and expanded, andten days later, subcloned into five 96-well plates. Two weeks later, theplates were screened for nucleolin reactivity, and T061509-9F9-1D11-1D10wells 2C7 and 2F8 were confirmed positive on the following day, and weresubcloned into 60 wells each on single 96-well plates. Ten days later,the plates were screened for nucleolin reactivity, andT061509-9F9-1D11-1D10-2F8 wells 2D11 and 2B6 had the highest reactivity,and were subcloned the next day into 60 wells each on single 96-wellplates.

6^(th) Round Subcloning.

Seventeen days later, the subclone plates were screened for nucleolinreactivity, and T061509-9F9-1D11-1D10-2F8-2D11 well 1F4 had the highestnucleolin reactivity, and was subcloned the next day by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweenweeks 4 and 6. Three wells (4E8, 1C7, 8A6) had reactivity. CloneT061509-9F9-1D11-1D10-2F8-2D11-1F4-4E8 was expanded for functionalantibody testing because of its superior growth properties.

Clone 8G4.

Initial Screening and Primary Subcloning.

A tonsil sample was received and processed into an immortalizedrepertoire consisting of 10×96-well plates (T081009). Eleven days later,the library was screened for reactivity to nucleolin. ELISA confirmationwas conducted, identifying reactive well (T081009-8G4), which wassubcloned into 60 wells on a single plate.

Secondary Subcloning.

The subclone plate was screened 19 days later and 2 wells wereidentified with nucleolin reactivity (T081009-8G4 wells 1C9 and 1F3).Well 1F3 had the highest reactivity and was subcloned into 60 wells on asingle 96-well plate 3 days later.

Tertiary Subcloning.

Three weeks later, the subclone plate was screened and multiple wellshad nucleolin reactivity. T081009-8G4-1F3 wells 2G4 and 2H3 had thehighest reactivity and were subcloned the next day into 60 wells each onsingle 96-well plates. The remainder of the reactive wells were pooled.Five days later, the subcloned plates showed evidence of fungalcontamination and were discarded. Reactive cells from the pooledremainder (T081009-8G4-1F3) were expanded and two weeks later subclonedinto ten 96-well plates. Thirteen days later these subclone plates werescreened, and T081009-8G4-1F3 wells 4G2 and 5B9 had the highestnucleolin reactivity and were subcloned two days later into 60 wellseach on single 96-well plates.

Quaternary Subcloning.

Three weeks later, the subclone plates were screened, andT081009-8G4-1F3-4G2 wells 1E9 and 2F5 had the highest nucleolinreactivity and were subcloned the next day into 60 wells each on single96-well plates.

5^(th) Round Subcloning.

Thirteen days later, the subclone plates were screened, andT081009-8G4-1F3-4G2-2F5 wells 2C10 and 2E8 had the highest nucleolinreactivity and were subcloned the next day into 60 wells each on single96-well plates.

6^(th) Round Subcloning.

Twenty-five days later, the subclone plates were screened, andT081009-8G4-1F3-4G2-2F5-2E8 well 2D11 had the highest nucleolinreactivity. Four days later, well 2D11 was subcloned by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweendays 17 and 33. Multiple wells (7E9, 4C9, 4D9, 6A3, 10F8, 8B3) hadreactivity. Clone T081009-8G4-1F3-4G2-2F5-2E8-2D11-8B3 was expanded forfunctional antibody testing because of its superior growth properties.

Clone P1C6.

Initial Screening and Primary Subcloning

A peripheral blood sample was received and processed into animmortalized repertoire consisting of 10×96-well plates (PB120909). Ninedays later, the library was screened for reactivity to nucleolin. ELISAconfirmation was conducted, identifying reactive well (PB120909-106),which was subcloned 3 days later into 60 wells on a single plate.

Secondary Subcloning.

The subclone plate was screened three weeks later, and 2 wells wereidentified with nucleolin reactivity (PB120909-1C6 wells 1H2 and 1H9).Both were subcloned into 60 wells each on single 96-well plates.

Tertiary Subcloning.

Seventeen days later, the subclone plates were screened andPB120909-1C6-1H9 wells 2F4 and 2G3 had the highest nucleolin reactivityand were subcloned into 60 wells each on single 96-well plates.

Quaternary Subcloning.

Three weeks later, the subclone plates were screened, andPB120909-1C6-1H9-2G3 wells 2E7 and 2C9 had the highest nucleolinreactivity and were subcloned into 60 wells each on single 96-wellplates.

5th Round Subcloning.

Seventeen days later, the subclone plates were screened, andPB120909-1C6-1H9-2G3-2C9 wells 1G6 and 1H10 had the highest nucleolinreactivity and were subcloned the next day into 60 wells each on single96-well plates.

6^(th) Round Subcloning.

Two weeks later, the subclone plates were screened, andPB120909-1C6-1H9-2G3-2C9-1G6 well 1F5 had the highest nucleolinreactivity. Three days later, well 1F5 was subcloned by limitingdilution cloning into ten 96-well plates. Clonal colonies appearedslowly over time, and were screened for nucleolin reactivity betweendays 22 and 28. Multiple wells (3C7, 7D6, 10F9, 10G10) had reactivity.Clone PB120909-1C6-1H9-2G3-2C9-1G6-1F5-3C7 was expanded for functionalantibody testing because of its superior growth properties.

Example 2 Results

Nucleolin and Bcl-2 Protein are Overexpressed in the Plasma Membrane AndCytoplasm of B-CLL Cells Compared to B Cells from Normal HumanVolunteers.

CLL is indolent during most of its clinical course and the clonal Bcells accumulate in the bone marrow and circulation during the indolentphase by avoiding apoptosis (Klein et al., 2000). CLL cells circumventapoptosis by over-expressing the anti-apoptotic protein Bcl-2.High-level expression of bcl-2 mRNA and protein is seen in the absenceof gene rearrangements that are known to enhance bcl-2 transcription(Bakhshi et al., 1985; Robertson et al., 1996; Steube et al., 1995). Oneof the inventors discovered that bcl-2 mRNA is highly stabilized in CLLcells from patients compared to normal CD19+ B cells from healthyvolunteers (Otake et al., 2007). In addition, the inventors showed thatthe enhanced stability of bcl-2 mRNA in CLL cells was a direct result ofbinding of the stabilizing protein nucleolin to an ARE element in the3′-UTR of bcl-2 mRNA. Furthermore, nucleolin was over-expressed in thecytoplasm of CLL cells from all of the patients examined (FIG. 1).

Peripheral blood samples were obtained from 17 patients with untreatedCLL and 9 healthy volunteers. Mononuclear cells were isolated from eachblood sample and the B cells were purified from this fraction byimmuno-magnetic separation using positive selection for CD19, a pan-Bcell marker. The cytosolic levels of nucleolin and Bcl-2 protein werethen compared in CD19+ CLL cells from the patients to the levels inCD19+ B cells from the healthy volunteers (Otake et al., 2007).Cytoplasmic levels of nucleolin were examined because that is postulatedto be the site of stabilization of bcl-2 mRNA. The non-nuclear(cytoplasmic) levels of nucleolin and Bcl-2 protein were determined byimmunoblots of S10 extracts. To accurately compare the immuno-blotresults from different patients, the integrated density values (IDV) ofthe nucleolin and Bcl-2 protein bands in the immunoblots were normalizedto the IDV values obtained from known amounts of nucleolin and Bcl-2external standards. This analysis revealed that Bcl-2 levels were11-fold elevated (p<0.001) and nucleolin levels were 26-fold elevated(p<0.001) in CLL cells from 17 patients compared to B cells from 9normal volunteers (FIG. 1). In addition, the enhanced Bcl-2 proteinlevels positively correlated with the increased nucleolin levels(Pearson's correlation=0.83, p<0.001). No significant difference wasobserved in the levels of nucleolin in nuclear fractions between CLL andnormal B cells. The fact that nucleolin was uniformly over-expressed inall of the CLL patients, including those in early stages of diseasewithout prior therapy, suggests that nucleolin stabilization of bcl-2mRNA is an early event in CLL pathogenesis, rather than a feature ofdisease evolution or an epiphenomenon of chemotherapy.

Nucleolin and Bcl-2 Protein are Overexpressed in the Plasma Membrane andCytoplasm of B-CLL Cells Compared to B Cells from Normal HumanVolunteers.

Confocal microscopy studies of CLL cells from patients and B cells fromhealthy volunteers were performed to confirm the results obtained byimmuno-blotting. The localization of nucleolin was determined byindirect immunofluorescence using anti-nucleolin MAb and aFITC-anti-mouse IgG secondary antibody. The DNA was stained withpropidium iodide. The overlay images in FIG. 2A indicate that nucleolinwas present in the plasma membrane, cytoplasm and nucleus of CLL cells,but was only present in the nucleus of B cells. These results wereconsistent with the immunoblots indicating localization of nucleolin inthe plasma membrane and cytoplasm of CLL cells but not in normal Bcells.

Nucleolin is Also Highly Overexpressed in MCF-7 Breast Cancer CellsCompared to MCF-10A Normal Mammary Epithelial Cells.

The intracellular localization of nucleolin in MCF-7 and MDA-MB-231breast cancer cells and MCF-10A normal mammary epithelial cells wasdetermined by indirect immunofluorescence as described above(Soundararajan et al., 2008). The overlay images in FIG. 2B indicatethat nucleolin was present throughout the nucleus, plasma membrane andcytoplasm (green fluorescence) of MCF-7 cells, while in normal MCF-10Acells nucleolin was detected only in the nucleus. This was also true forMDA-MB-231 breast cancer cells (Soundararajan et al., 2008)

Nucleolin and Bcl-2 Protein are Also Overexpressed in the PlasmaMembrane and Cytoplasm of MCF-7 and MDA-Mb-231 Breast Cancer CellsCompared to MCF-10A Normal Mammary Epithelial Cells.

The intracellular localization of nucleolin was determined by indirectimmunofluorescence using primary antibody against nucleolin and aFITC-conjugated anti-mouse IgG secondary antibody (green fluorescence).The DNA was stained with propidium iodide (red fluorescence). Theoverlay images in FIG. 3 indicate that nucleolin was present throughoutthe nucleus (yellow fluorescence), plasma membrane and cytoplasm (greenfluorescence) of MCF-7 cells, while in normal MCF-10A cells nucleolinwas detected only in the nucleus. The intracellular localization ofnucleolin in MDA-MB-231 breast cancer cells (Soundararajan et al., 2008)was similar to that of MCF-7 cells.

AML Blast Cells from Patients that Engraft in NOD/SCID Mice Show IntenseNucleolin Staining.

Further evidence that nucleolin is a tumor-specific antigen and also hasa role in human AML pathogenesis comes from human AML xenograft studies.Biopsy specimens from the bone marrow were assessed for expression ofnucleolin in the blast cytoplasm and/or nucleus. Immunohistochemicalanalysis was performed on glass slides of paraffin-embedded tissuesections of archived bone marrow biopsies. In all cases, the AML blastswere compared to the surrounding normal bone marrow elements. Theinventors observed that anti-nucleolin staining was intense in the AMLblast cells (fuchsin stain), while normal elements of the bone marrow(dark blue counterstain) were either weakly positive or outrightnegative (FIG. 4). Strong expression of nucleolin has been observed inall AML samples tested to date.

Ten NOD-SCID male mice (eleven weeks of age) were each injected i.p.with 25 μg of anti-asialo GM antibody and exposed to 2.3 Gy in a Cs137irradiator. The same day, five of the ten mice were each injected i.p.with 2×10⁷ PBMC from a human subject diagnosed with AML. At least 60% ofthe PBMC in this subject were AML blasts. Four months after the i.p.injection of PBMC all mice were euthanized. In two of five mice injectedi.p. with AML cells, the spleens were enlarged and showed nodularlesions on the surface of the spleen as well as within it. Part of thespleen of each mouse was processed to dissociate splenocytes andanalyzed by flow cytometry for the presence of human CD45+ cells. Inmice with abnormal spleens, some of the spleenocytes were stained withanti-human CD45 antibodies, demonstrating that these mice had beenengrafted with human AML cells. The inventors next carried outhistopathology and immunohistochemistry analyses on the spleen ofengrafted and control NOD/SCID mice (FIG. 5A). Nodular areas of leukemicengraftment in the spleen were strikingly positive for nucleolinexpression.

Nucleolin is Also Highly Overexpressed in Human Breast CarcinomasCompared to Normal Mammary Tissue.

Furthermore, nucleolin immunohistochemistry performed on a human breastcarcinoma tissue array comprising tumor and normal adjacent breasttissue from 50 patients indicated that nucleolin was overexpressed inthe tumors compared to the normal adjacent tissue. Representativeresults are depicted for the most common forms of breast cancer (FIG.5B). Nucleolin was highly expressed in invasive ductal carcinoma (8/8cases tested) and medullary carcinoma (6/6 cases), and was moderatelyexpressed in mucinous adenocarcinoma (8/8 cases), but was not detectedin adjacent normal tissue (FIG. 5B).

The results presented in FIGS. 1-5B above provide strong evidence thatnucleolin is overexpressed on the cell surface of certain human tumorcells as a tumor-specific antigen. For human CLL (and possibly AML andbreast cancer), nucleolin overexpression is an early event in thepathogenesis of this disease. The nucleolin-targeting aptamer AS1411,which is considered a ‘chemical’ anti-nucleolin antibody (Soundararajanet al., 2008; Soundararajan et al., 2009) showed promising activity inphase II clinical trials for refractory and relapsed AML (ClinicalTrials.gov Identifier NCT00512083). Thus, when taken together theseresults indicate that nucleolin is an excellent target for thedevelopment of therapeutic antibodies.

Bcl-2 mRNA Stability is Increased in CLL Cells Relative to Normal BcCells.

The overexpression of Bcl-2 in CLL cells compared to normal B cellscould result either from enhanced bcl-2 mRNA transcription, increasedbcl-2 mRNA stability, or increased efficiency of bcl-2 mRNA translation.It is difficult to measure mRNA stability in primary CLL cells with thestandard method using actinomycin D to block transcription, since bcl-2mRNA is very stable in CLL cells requiring long incubation times withactinomycin D, which is toxic to the cells. To circumvent this problem,the inventors measured the levels of nascent, unspliced heterogeneousnuclear bcl-2 mRNA (hnRNA) and mature bcl-2 mRNA in CLL cells and normalB cells from healthy volunteers. This method has been used successfullyto determine the relative rate of mRNA transcription and mRNA decay in avariety of cells. Equal amounts of total RNA from each sample werereverse-transcribed and real-time PCR was performed with two sets ofprimers. One reaction contained primers that anneal to the first intron(to selectively amplify hnRNA) and one with primers that anneal tosequences in two adjacent exons (to selectively amplify spliced, matureRNA). The inventors found that the ratio of bcl-2 mRNA to bcl-2 hnRNAwas about 3-fold higher for CLL cells compared to normal B cells(p<0.001) (FIG. 6). The 3-fold higher ratio of bcl-2 mRNA/bcl-2 hnRNAfor CLL cells was entirely due to increased levels of bcl-2 mRNA in CLLcells (3.3±0.4 SEM relative to βl-actin mRNA) compared to bcl-2 mRNAlevels in normal B cells (1.1±0.2 SEM relative to β-actin mRNA). Nosignificant difference was observed in the level of bcl-2 hnRNA in CLLcells (6.5±1.4 SEM relative to βl-actin mRNA) versus normal B cells(5.5±1.4 SEM relative to β-actin mRNA). These results indicate thatbcl-2 mRNA is relatively more stable in CLL cells compared to normal Bcells. Furthermore, the rate of bcl-2 mRNA transcription was notrelatively higher in CLL cells because the bcl-2 mRNA/hnRNA ratio wouldhave been lower in CLL versus normal B cells if that were true.

Mechanism by which Nucleolin Overexpression Upregulates Bcl-2 mRNA.

The mechanism of bcl-2 mRNA stabilization by nucleolin was examined byfollowing the decay rate of bcl-2 RNA transcripts in extracts preparedfrom purified CLL cells and normal B cells, using an in vitro RNA decaysystem (Sengupta et al., 2004). Capped and polyadenylated mRNAs wereused in these assays to mimic in vivo decay, which involvescap-stimulated deadenylation by poly (A)-specific ribonuclease (PARN),followed by rapid decay of the mRNA body by the exosome (Chen et al.,2001; Mukherjee et al., 2002). ³²P-labeled bcl-2-ARE transcripts wereincubated with cytoplasmic S100 extracts from CLL and normal B cells inthe presence of poly(A) to activate deadenylation. As shown in FIG. 7,bcl-2 transcripts decayed more rapidly in extracts of normal B cellsthan in extracts of CLL cells. The average half-life of bcl-2 RNA incytoplasmic extracts of CLL cells from 4 patients was estimated to be 72min by extrapolation of the data, while the average half-life of thetranscript in normal B cell extracts was 12 min (FIG. 7). The rapiddecay of the bcl-2-CR (coding region)-ARE RNA transcripts in normal Bcell extracts was highly ARE-dependent, since the rates of decay ofbcl-2 mRNA coding region transcripts lacking the ARE (bcl-2-CR RNA) weresimilar in normal B cell and CLL cell extracts. It is also important tonote that addition of 280 nM purified recombinant nucleolin [Δ1-283Nuc-(His)6] to extracts of normal B cells greatly slowed the decay rateof bcl-2-ARE (extrapolated half-life of 62 min); When taken together,these results indicate that nucleolin stabilizes bcl-2 mRNA by bindingto an ARE in the 3′-UTR of bcl-2 mRNA and protecting the mRNA fromdegradation.

Development of Nucleolin-Specific Human Antibodies.

Cumulatively, the preliminary studies described above indicate thatnucleolin is a promising tumor antigen target for antibody basedimmunotherapy of CLL and AML, and possibly for certain forms of breastcancer. The inventors therefore set out to create therapeutic human MAbsspecific for nucleolin, using a novel in vitro method for producinghuman MAbs from immortalized B cell libraries created from human tonsil.The technology holds several advantages including 1) rapid antibodyproduction with all steps in vitro; 2) human immunization is notrequired, 3) the resulting antibodies are fully human and thereforeshould not be rejected, nor cause serum sickness. This technology wasinvented with support of the National Cancer Institute and the FederalGovernment has certain rights in the invention. A worldwide patentapplication (PCT/US2008/072124) was filed in August, 2008. An overviewof the technology is depicted in FIG. 8. Human B cells were isolatedfrom tonsillectomy specimens from healthy children, then efficientlyinfected with Epstein-Barr virus (EBV) resulting in wide scale B celltransformation. Infected B cells were induced to differentiate withagents that mimic B cell receptor signaling and T cell help. Sincetonsil is a rich source of B cells, this process typically allowed forthe isolation of 10⁷-10⁸ B cells, each with unique antibody specificity.The efficient infection process is demonstrated in FIG. 9A, which showsthat after spinfection of tonsil B cells with 10× concentrated viralstocks of recombinant EBV containing a green fluorescent protein markergene (EBfaV-GFP (Speck et al., 1999a; Speck et al., 1999b), nearly 100%of the tonsil B cells were infected after 24 h. The infected cells werethen plated into 96-well plates at 10³-10⁴ cells per well in thepresence of a proprietary differentiation cocktail (Diff-Cktl)consisting of recombinant CD40L, Baff and anti-human IgM (Fab′)₂, thatinduced immunoglobulin (Ig) isotype class switching from IgM to IgG overa 2 week period. As can be seen in FIG. 9B, IgG was secreted into theculture supernatant at levels typically ranging from 1-4 μg/ml. Thisprocess reproducibly creates EBV immortalized tonsil B cell librariessecreting polyclonal IgG with an estimated 10⁶-10⁷ antibodyspecificities, depending upon the number of B cells isolated from anindividual tonsil. Cell culture supernatants from the libraries can thenbe screened for specific binding to various antigens of interest. Bcells producing IgG with the desired specificity can then be isolated bylimiting dilution cloning, and the IgG purified from the culturesupernatant. For larger scale production, recombinant IgG can beproduced by subcloning the clonal B cells' Ig genes into mammalianexpression vectors and transducing cell lines commonly used for theproduction of biologicals. To date, the inventors have created more than40 libraries (data not shown), which have been screened for reactivityto various antigenic targets.

This platform technology is ideal for producing autoantibodies withtherapeutic implications, such as antibodies to tumor antigens likenucleolin. Ordinarily, healthy children would not be expected to makestrong secondary antibody responses to self-proteins such as nucleolin,because autoreactive B cells do not normally receive sufficient T cellhelp required for their survival and differentiation into IgG secretingplasma cells. Instead, the autoreactive B cells become tolerized or aredeleted in vivo. However, these data indicate that it is possible torescue rare nucleolin reactive B cells from tonsil or peripheral blood,using EBV to immortalize the autoreactive B cells, and thedifferentiation cocktail to artificially force them to undergo Igisotype class switching and secretion of nucleolin specific IgG invitro. Using this approach, the inventors screened twelve immortalizedtonsil libraries for nucleolin specific IgG by enzyme linkedimmunosorbent assay (ELISA). As can be seen in FIG. 10, six human B celllines have been isolated that produce IgG antibodies that react stronglywith recombinant human nucleolin (Δ1-283Nuc-(His)₆) produced inbacteria. As a positive control, binding was compared to that obtainedwith mouse anti-nucleolin MAb MS3 (Santa Cruz Biotech), while culturesupernatant from a human B cell line raised in the same manner, butproducing IgG1 antibodies specific for H5 hemagglutinin (human anti-HA)served as a negative control.

Preliminary Characterization of Human Anti-Nucleolin Antibodies.

In order to test whether any or all of these anti-nucleolin antibodiesmight have therapeutic potential, the inventors have beguncharacterizing the antibodies by further ELISA analyses, Westernblotting, flow cytometry, immunohistochemistry and cytotoxic killing ofMV4-11 cells. Four of the antibodies were initially screened further byELISA in order to test whether they recognize endogenous nucleolinpurified from MV4-11 human leukemia cells, and to recombinant His-taggednucleolin (Δ1-283 Nuc-(His)₆ produced in bacteria; Ishimaru et al.,2009). As can be seen in FIG. 11, top panel, supernatants from three outof four of the B cell lines (5D1, 7G7 and 2D3) reacted with endogenousnucleolin with similar (or higher) intensity as mouse anti-nucleolinMS3, while all bound the his-tagged recombinant to varying degrees. Inaddition, three of the four culture supernatants (5D1, 3H11, and 2D3)reacted to the his-tagged recombinant nucleolin by Western blotting(FIG. 11, bottom). Thus, the antibodies had distinct patterns of bindingto the endogenous and his-tagged proteins, and reacted differently inthe Western blotting analysis, possibly indicating that each recognizesdistinct protein epitopes.

Anti-Nucleolin Cytotoxicity to MV4-11 AML Cells and MCF-7 Breast CancerCells in the Absence of CDCC or ADCC.

FIG. 12 further demonstrates that all of the anti-NUC HuMAbs bind tocell surface nucleolin on MV4-11 AML cells, but they do not bind tonormal tonsil cells, which do not express nucleolin on the cell surface.In contrast, Rituximab bound to normal tonsil cells but not to MV4-11cells. Furthermore, the anti-NUC HuMAbs were cytotoxic to MV4-11 cells(FIG. 14C). After incubation of MV4-11 cells with each antibody (2μg/ml), mitochondrial function was measured by MTS assay at 48, 72 and96 h. FIG. 14C demonstrates that by 96 h, cell viability decreased inresponse to each antibody, ranging from ˜30-80% of untreated cells. Bycomparison, after incubation with nucleolin-targeting aptamer AS1411 (20μM), cell viability decreased to ˜40-60% of untreated cells, and wasunchanged by incubation with control human IgG. This assay was performedin the absence of complement and without the effector cells required forADCC, indicating that the anti-nucleolin HuMAbs acted independently ofCDCC and ADCC. A summary of these findings is presented in Table 4.Taken together, these data provide strong evidence that each of the sixantibodies isolated have distinct binding characteristics and are eitherbinding to different epitopes on human nucleolin, and/or have distinctaffinities for the protein.

While the binding analysis has fundamental significance, from atherapeutic standpoint the most important criterion is that theantibodies mediate cytotoxicity. For this reason, the inventors set up asimple flow cytometric based assay measuring induction of apoptosisand/or cell death by propidium iodide (PI) and annexin-V staining.Annexin-V binds to phoshatidyl serine residues, which become exposed onthe cell membrane as an early event during induction of apoptosis. Atlater stages of apoptosis or during necrotic cell death, disruption ofmembrane integrity makes the cells permeable to the non-vital dye PI,which can be detected through flow cytometry. MV4-11 cells were culturedfor 16 h in the presence of each antibody and human serum, which wasused as a source of complement and is required for detecting complementdependent cellular cytotoxicity (CDCC), after which they were stainedwith PI and annexin V-FITC. As can be seen in FIG. 13, MV4-11 cellstreated with human anti-HA antibodies (negative control) showed nosignificant differences in either annexin-V or PI staining compared withuntreated cells. In contrast, each of the anti-nucleolin antibodiesinduced about a 3-fold shift in the mean fluorescence intensity ofannexin-V staining compared to untreated or anti-HA treated cells. Asimilar shift was also seen in cells treated with staurosporine (25 μM),a chemical inducer of apoptosis. Anti-nucleolin antibodies 5D1 and 7G7were the most cytotoxic antibodies, with 38-40% of cells stainingpositive for PI and/or annexin-V after 16 h, compared to just 16% forcontrol anti-HA treatment, while the other anti-nucleolin antibodiesinduced cytotoxicity in ˜30% of cells. The relative cytotoxic activitiesof each antibody against MV4-11 leukemia cells are summarized in Table4. These preliminary data indicate that more comprehensive cytotoxicitytesting is warranted, particularly for antibodies 5D1 and 7G7.

Similar experiments were performed with MCF-7 breast carcinoma cells,which were found to be more sensitive to the anti-nucleolin HuMAbs thanwere the MV4-11 cells. In the absence of complement, the anti-nucleolinHuMabs inhibited cell viability to less than 20% of untreated cellsafter 96 h, while AS1411 reduced cell viability to just 80% of untreatedcells, and control human IgG had no effect (FIG. 14A, left panel).Complement dependent cytotoxicity was assayed in parallel experimentswith the addition of human AB serum (25% vol/vol) as a source ofcomplement. As can be seen in FIG. 14A, right panel, the addition ofcomplement potentiated the cytotoxic effects of the antibodies,significantly inhibiting cell viability as early as 48 h, with completeinhibition by 96 h. FIG. 14B shows that the cytotoxic effects wereselective for MCF-7 cells, since the HuMAbs had no effect on cellviability of MCF-10A normal mammary epithelial cells that do not expresscell surface nucleolin. Thus, our panel of anti-nucleolin HuMAbs bindspecifically to cell surface nucleolin expressed on human AML and breastcancer cell lines and can induce substantial tumor cell cytotoxicitythat is independent of the immune mechanisms of ADCC and CDCC. but canbe potentiated by serum complement.

TABLE 4 Summary of Results Performed on Characterization of HumanAnti-Nucleolin Antibodies ELISA Cytotoxicity Hu-anti Recombinant^(a)/MV4-11^(c)/ Heavy Light Nuc Endogenous^(b) Western FACS MCF-7^(d) ChainChain IHC 2D3 +++ ++ ++ ++ ++ ++++ IgG1 Lambda n.d. 2H3 +++ n.d. n.d. +++ +++ IgG1 Kappa n.d. 3H11 ++++ — ++ ++ ++ ++++ IgG1 Lambda n.d. 5D1+++ + ++ ++++ +++ ++++ IgG1 Kappa + 7G7 +++ +++ — ++ +++ +++ IgG1 Kappan.d. 9F9 ++++ n.d. n.d. ++ ++ ++++ IgG1 Kappa + 8G4 +++ n.d. n.d. ++++++ ++++ IgG1 Lambda n.d. P1C6 +++ n.d. n.d. +++ +++ ++++ IgG1 Kappan.d. ^(a)Binding to recombinant 1-283Nuc-(His)₆ nucleolin; ^(b)bindingto nucleolin purified from MV4-11 leukemia cells; ^(c)cytotoxicityagainst MV4-11 cells measured by MTS assay at 96 h; ^(d)cytotoxicityagainst MCF-7 cells measured by MTS assay at 96 h; n.d.: not done.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations can be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

VIII. REFERENCES

The following references, and any others cited throughout theapplication, to the extent that they provide exemplary procedural orother details supplementary to those set forth herein, are specificallyincorporated herein by reference.

-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,179,337-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,301,144-   U.S. Pat. No. 4,366,241-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,485,045-   U.S. Pat. No. 4,496,689-   U.S. Pat. No. 4,544,545.-   U.S. Pat. No. 4,640,835-   U.S. Pat. No. 4,657,760-   U.S. Pat. No. 4,670,417-   U.S. Pat. No. 4,791,192-   U.S. Pat. No. 4,816,567-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 4,975,278-   U.S. Pat. No. 5,013,556-   U.S. Pat. No. 5,021,236-   U.S. Pat. No. 5,047,335-   U.S. Pat. No. 5,196,066-   U.S. Pat. No. 5,206,344-   U.S. Pat. No. 5,225,212-   U.S. Pat. No. 5,278,299-   U.S. Pat. No. 5,510,261-   U.S. Pat. No. 5,534,615-   U.S. patent application Ser. No. 12/671,936-   U.S. Patent Publn. 2009/0191244-   U.S. Patent Publn. 2007/0066554-   U.S. Patent Publn. 2007/0066554-   Atherton et al., Biol. Reprod., 32(1):155-171, 1985.-   Bakhshi et al., Cell, 41(3):899-906, 1985.-   Bates et al., Exp. Mol. Path., 86:151, 2009.-   Bose et al., J. Virol., 78:8146, 2004.-   Boyd et al., Mol. Immunol., 32:1311-1318, 1996.-   Chen et al., Cell, 107(4):451-64, 2001.-   Chen et al., Genes Dev., 14(10):1236-48, 2000.-   Chen et al., Mol. Ther., 16(2):333-42, 2008.-   Christian et al., J. Cell Biol., 163:871-878, 2003.-   Cunningham and Wells, Science, 244:1081-1085, 1989.-   Destouches et al., PLoS ONE, 3(6):e2518, 2008.-   Dholakia et al., J. Biol. Chem., 264(34):20638-20642, 1989.-   Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688, 1985.-   Evan et al., Molec. Cell. Biol. 5:3610-3616, 1985.-   Farin et al., PLoS ONE, 4(7): 6128, 2009.-   Field et al., Mol. Cell. Biol., 8:2159-2165, 1988.-   Fogal et al., Angiogenesis, 12(1):91-100, 2009.-   Gabizon et al., J. National Cancer Inst., 81(19):1484, 1989.-   Gattoni-Celli et al., Am. J. Hematol., 84(8):535-8, 2009.-   Geahlen et al., Biochim. Biophys. Acta., 804:169-175. 1984.-   Ginisty et al., J. Biol. Chem., 276(17):14338-43, 2001.-   Hopp et al., BioTechnology, 6:1204-1210, 1988.-   Hovanessian et al., Cell Research, 16:174-181, 2006.-   Hovanessian et al., Exp. Cell Res., 261(2):312-28, 2000.-   Hse et al., J. Biol. Chem., 272:9062-9070, 1997.-   Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030, 1980.-   Ishimaru et al., Mol. Cancer. Res., 7(8):1354-66, 2009.-   Izumi et al., Viral Research, 76(1):17-29, 2001.-   Jefferis and Lund, Chem. Immunol., 65:111-128, 1997.-   Jefferis and Lund, Current Opin. Biotech., 7:409-416, 1996.-   Khatoon et al., Ann. Neurol, 26(2):210-215, 1989.-   Kibbey et al., J. Neurosci. Res., 42(3):314-22, 1995.-   Kim and Baldwin, Ann. Rev. Biochem., 51:459-89, 1982.-   King et al., J. Biol. Chem., 264(17):10210-10218, 1989.-   Klein et al., Leukemia, 14(1):40-6, 2000.-   Lapeyre et al., Proc. Natl. Acad. Sci. USA, 84(6):1472-6, 1987.-   Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397,    1990.-   Malhotra et al., Nature Med., 1:237-243, 1995.-   Martin et al., J. Biol. Chem., 257:286-288, 1982.-   Martin et al., Science, 255:192-194, 1992.-   Massey, Nature, 328:457-458, 1987.-   Miller and Lipman, Proc. Natl. Acad. Sci. USA, 70:190-194, 1973.-   Mukherjee et al., Embo. J., 21(1-2):165-74, 2002.-   Neuberger et al., Nature, 312:604-608, 1984.-   O'Shannessy et al., Anal. Biochem., 163(1):204-9, 1987.-   Otake et al., Blood, 109(7):3069-75, 2007.-   Owens and Haley, Biochem. Biophys. Re.s Commun., 142(3):964-971,    1987.-   Paborsky et al., Protein Engineering, 3(6):547-553, 1990.-   PCT Appln. PCT/US2008/072124-   PCT Appln. WO 81/01145-   PCT Appln. WO 88/07378-   PCT Appln. WO 93/23572-   PCT Appln. WO 94/11026-   PCT Appln. WO 96/32478-   Pfeifle and Anderer, Biochim. Biophys. Acta, 762(1):86-93, 1983.-   Potter and Haley, Methods Enzymol, 91:613-633, 1983.-   Remington's Pharmaceutical Sci., Maack Publishing Co, Easton Pa.,    1990.-   Robertson et al., Leukemia, 10(3):456-9, 1996.-   Said et al., J. Biol. Chem., 277(40):37492-502, 2002.-   Sengupta et al., J. Biol. Chem., 279(12):10855-63, 2004.-   Serin et al., J. Biol. Chem., 272(20):13109-16, 1997.-   Sinclair and O'Brien, J. Biol. Chem., 277(4):2876-85, 2002.-   Skinner et al., J. Biol. Chem., 266:15163-15166, 1991.-   Soundararajan et al., Cancer Res., 68(7):2358-65, 2008.-   Soundararajan et al., Mol. Pharmacol., 76(5):984-91, 2009.-   Speck et al., Arch. Virol., 144(6):1123-37, 1999b.-   Speck et al., J. Gen. Virol., 80 (Pt 8):2193-2203, 1999a.-   Srivastava and Pollard, Faseb J., 13(14):1911-22, 1999.-   Srivastava et al., FEBS Lett., 250(1):99-105, 1989.-   Steube et al., Leukemia, 9(11):1841-6, 1995.-   Takekoshi et al., J. Biochem., 130:299-303, 2001.-   Umana et al. Mature Biotech., 17:176-180, 1999.-   Vitetta et al., Science, 238:1098, 1987.-   Weltschof et al., J. Immunol. Methods, 179:203-214, 1995.-   Westmark and Malter, Brain Res. Mol. Brain. Res., 90(2):193-201,    2001.-   Wittwe and Howard, Biochem., 29:4175-4180, 1990.-   Wright and Morrison, Trends Biotechnol., 15:26-32, 1997).-   Yang et al., Nucl Acids Res., 30:2251-2260, 2002.

The invention claimed is:
 1. A cytotoxic isolated human or humanizedanti-nucleolin antibody, or fragment thereof, that binds to amino acidsequence SEQ ID NO:4 or the amino acid sequence encoded by DNA sequenceSEQ ID NO:3, wherein the antibody, or fragment thereof, kills a cancercell that expresses cell surface nucleolin.
 2. The cytotoxic isolatedhuman or humanized anti-nucleolin antibody, or fragment thereof, ofclaim 1, wherein the antibody, or fragment thereof, kills at least 10%of a population of MCF-7 or MV 4-11 cells, when incubated with the MCF-7or MV 4-11 cells and human AB serum for 48 hours or less.
 3. Thecytotoxic isolated human or humanized anti-nucleolin antibody, orfragment thereof, of claim 1, wherein the antibody or fragment thereofis a Fab, Fab′, F(ab′)2, or Fv fragment; a diabody; a linear antibody; asingle-chain antibody; or a multispecific antibody formed from anantibody fragment.
 4. The cytotoxic isolated human or humanizedanti-nucleolin antibody, or fragment thereof, of claim 1, furthercomprising a radionuclide, a fluorophore, a chemilluminescent compound,a fluorescent compound, an enzyme, a toxin, or a chemotherapeutic agent.5. A method of producing an immortalized human B-cell that secrets acytotoxic human or humanized antibody, or fragment thereof, that bindsto amino acid sequence SEQ ID NO:4 or the amino acid sequence encoded byDNA sequence SEQ ID NO:3, wherein the antibody, or fragment thereof,kills a cancer cell that expresses cell surface nucleolin, the methodcomprising the steps of: (a) contacting a population of IgM-positivehuman B-cells with: (i) Epstein-Barr virus (EBV), and (ii) acytokine/growth factor/signaling agent cocktail; and (b) culturing humanB-cells from (a) under conditions supporting immortalization of thehuman B-cells and induction of IgM-to-IgG immunoglobulin isotypeclass-switching of the human B-cells, wherein steps (a) and (b) areperformed ex vivo.
 6. The method of claim 5, wherein the cytokine/growthfactor/signaling agent cocktail comprises an agent that delivers acostimulatory signal to a human B-cell.
 7. The method of claim 5,wherein the cytokine/growth factor/signaling agent cocktail comprisesanti IgM F(ab′)₂, interleukin(IL)-2, IL-4, IL-5, IL-6, IL-9, IL-10,IL-13, INFα, BAFF, soluble CD40L, or any combination thereof.
 8. Amethod of killing a cell that expresses nucleolin on its surface,comprising contacting the cell that expresses nucleolin on its surfacewith an effective amount of a cytotoxic isolated human or humanizedanti-nucleolin antibody, or fragment thereof, to kill the cell thatexpresses nucleolin on its surface, wherein the antibody, or fragmentthereof, binds to amino acid sequence SEQ ID NO: 4 or the amino acidsequence encoded by DNA sequence SEQ ID NO:3, wherein the antibody, orfragment thereof, kills a cancer cell that expresses cell surfacenucleolin.
 9. The method of claim 8, wherein the cell that expressesnucleolin on its surface is selected from the group consisting of: alung cancer cell, a breast cancer cell, a prostate cancer cell, a coloncancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, anovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastomacell, a neuroblastoma cell, a sarcoma cell, and a gastric cancer cell.10. The method of claim 8, further comprising contacting the cell thatexpresses nucleolin on its surface with at least one additionalinhibitory agent or additional treatment.
 11. The method of claim 10,comprising contacting the cell that expresses nucleolin on its surfacewith the additional treatment, wherein the additional treatmentcomprises surgery, radiotherapy, chemotherapy, toxin therapy,immunotherapy, hormone therapy, anti-angiogenic therapy, gene therapy,other biological therapies, or any combination thereof.
 12. The methodof claim 10, comprising contacting the cell that expresses nucleolin onits surface with the additional inhibitory agent, wherein the additionalinhibitory agent comprises a radionucleotide, chemotherapeutic agent,toxin, immunotherapeutic, hormone, nucleic acid, polypeptide, or anycombination thereof.
 13. The method of claim 12, wherein the additionalinhibitory agent comprises the toxin, and wherein the toxin is selectedfrom the group consisting of: diphtheria toxin, exotoxin A chain, ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, phytolaca americana protein, pokeweedantiviral protein, momordica charantia inhibitor, curcin, crotin,sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,phenomycin, enomycin, calicheamicins, and tricothecenes toxin.
 14. Themethod of claim 12, wherein the additional inhibitory agent comprisesthe chemotherapeutic agent, and wherein the chemotherapeutic agent isselected from the group consisting of: an alkylating agent,anthracycline, a cytoskeletal disruptor, epothilone, an inhibitor oftopoisomerase I, an inhibitor of topoisomerase II, a nucleoside ornucleotide analog, a precursor analog, a peptide antibiotic, aplatinum-based agent, a retinoid, a vinca alkaloid, and derivativesthereof.
 15. The cytotoxic isolated human or humanized anti-nucleolinantibody, or fragment thereof, of claim 1, which is an IgG or IgMantibody or fragment thereof.
 16. The cytotoxic isolated human orhumanized anti-nucleolin antibody, or fragment thereof, of claim 1,wherein the antibody has a reduced immunogenicity to a human compared toa corresponding non-human or non-humanized antibody.
 17. The cytotoxicisolated human or humanized anti-nucleolin antibody, or fragmentthereof, of claim 1, which is an IgG1, IgG2, IgG3, or IgG4 antibody orfragment thereof.
 18. The cytotoxic isolated human or humanizedanti-nucleolin antibody, or fragment thereof, of claim 1, wherein theantibody or fragment thereof comprises a kappa light chain.
 19. Animmortalized human B-cell expressing the cytotoxic isolated human orhumanized anti-nucleolin antibody, or fragment thereof, of claim
 1. 20.The immortalized human B-cell of claim 19, wherein the B-cell is anEBV-immortalized B-cell.
 21. The immortalized human B-cell of claim 19,the B-cell deposited as American Type Culture Collection (ATCC) DepositNo. PTA-11493, No. PTA-11495, PTA-11490, PTA-11496, PTA-11491,PTA-11492, PTA-11497, or PTA-11494.
 22. The immortalized human B-cell ofclaim 21, the B-cell deposited as American Type Culture Collection(ATCC) Deposit No. PTA-11497.
 23. The cytotoxic isolated human orhumanized anti-nucleolin antibody, or fragment thereof, of claim 1,wherein the cancer cell is selected from the group consisting of a lungcancer cell, a breast cancer cell, a prostate cancer cell, a coloncancer cell, a pancreatic cancer cell, a renal cell carcinoma cell, anovarian cancer cell, a leukemia cell, a melanoma cell, a glioblastomacell, a neuroblastoma cell, a sarcoma cell, and a gastric cancer cell.24. The cytotoxic isolated human or humanized anti-nucleolin antibody,or fragment thereof, of claim 1, wherein the cancer cell is an AMLcancer cell, a CLL cancer cell, or a breast cancer cell.
 25. Thecytotoxic isolated human or humanized anti-nucleolin antibody, orfragment thereof, of claim 1, which is a monoclonal antibody or fragmentthereof.
 26. The cytotoxic isolated human or humanized anti-nucleolinantibody, or fragment thereof, of claim 1, which exhibitscomplement-dependent cytotoxicity to a cancer cell.
 27. The cytotoxicisolated human or humanized anti-nucleolin antibody, or fragmentthereof, of claim 1, which induces apoptosis in a cancer cell.
 28. Thecytotoxic isolated human or humanized anti-nucleolin antibody, orfragment thereof, of claim 1, which reduces BCL-2 levels in a cancercell.
 29. The cytotoxic isolated human or humanized anti-nucleolinantibody, or fragment thereof, of claim 1, wherein the antibody orfragment thereof comprises a lambda light chain.
 30. The cytotoxicisolated human or humanized anti-nucleolin antibody, or fragmentthereof, of claim 1, wherein the antibody, or fragment thereof, killsmore MCF-7 breast cancer cells than MCF10A normal mammary epithelialcells when incubated with separate populations of MCF-7 cells and MCF10Acells and heat inactivated serum for 72 hours or 96 hours.
 31. Thecytotoxic isolated human or humanized anti-nucleolin antibody, orfragment thereof, of claim 1, wherein the antibody, or fragment thereof,kills more MCF-7 breast cancer cells than MCF10A normal mammaryepithelial cells when incubated with separate populations of MCF-7 cellsand MCF10A cells and human AB serum for 96 hours.
 32. The method ofclaim 12, wherein the additional inhibitory agent comprises thechemotherapeutic agent, and wherein the chemotherapeutic agent isactinomycin-D, all-trans retinoic acid, azacitidine, adriamycin,azathioprine, bleomycin, camptothecin, carboplatin, capecitabine,cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin,docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone,etoposide, fluorouracil, 5-fluorouracil (5FU), gemcitabine, hydroxyurea,hydrogen peroxide, idarubicin, imatinib, mechlorethamine,mercaptopurine, methotrexate, mitomycin C, mitoxantrone, oxaliplatin,paclitaxel, pemetrexed, teniposide, tioguanine, valrubicin, vinblastine,vincristine, vindesine, or vinorelbine.